Enzymes with ruvc domains

ABSTRACT

The present disclosure provides for endonuclease enzymes having distinguishing domain features, as well as methods of using such enzymes or variants thereof.

CROSS-REFERENCE

This application is a continuation of U.S. application Ser. No.16/917,837, filed on Jun. 30, 2020, which is a continuation in part ofInternational Application No. PCT/US2020/018432, filed on Feb. 14, 2020,entitled “ENZYMES WITH RUVC DOMAINS”, which application claims thebenefit of U.S. Provisional Application Nos. 62/874,414 filed on Jul.15, 2019, 62/805,899 filed on Feb. 14, 2019, 62/805,868 filed on Feb.14, 2019, and 62/805,878 filed on Feb. 14, 2019. This application alsoclaims the benefit of U.S. Provisional Application No. 63/022,320, filedon May 8, 2020, entitled “ENZYMES WITH RUVC DOMAINS”. All of theseapplications are incorporated by reference herein in their entiretiesBACKGROUND

Cas enzymes along with their associated Clustered Regularly InterspacedShort Palindromic Repeats (CRISPR) guide ribonucleic acids (RNAs) appearto be a pervasive (˜45% of bacteria, ˜84% of archaea) component ofprokaryotic immune systems, serving to protect such microorganismsagainst non-self nucleic acids, such as infectious viruses and plasmidsby CRISPR-RNA guided nucleic acid cleavage. While the deoxyribonucleicacid (DNA) elements encoding CRISPR RNA elements may be relativelyconserved in structure and length, their CRISPR-associated (Cas)proteins are highly diverse, containing a wide variety of nucleicacid-interacting domains. While CRISPR DNA elements have been observedas early as 1987, the programmable endonuclease cleavage ability ofCRISPR/Cas complexes has only been recognized relatively recently,leading to the use of recombinant CRISPR/Cas systems in diverse DNAmanipulation and gene editing applications.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 30, 2020, isnamed 55921_703_301_SL.txt and is 23,363,499 bytes in size.

SUMMARY

In some aspects, the present disclosure provides for an engineerednuclease system, comprising: (a) an endonuclease comprising a RuvC_IIIdomain and an HNH domain, wherein the endonuclease is derived from anuncultivated microorganism, wherein the endonuclease is a class 2, typeII Cas endonuclease; and (b) an engineered guide ribonucleic acidstructure configured to form a complex with the endonuclease comprising:(i) a guide ribonucleic acid sequence configured to hybridize to atarget deoxyribonucleic acid sequence; and (ii) a tracr ribonucleic acidsequence configured to bind to the endonuclease. In some embodiments,the RuvC_III domain comprises a sequence with at least 70%, at least75%, at least 80% or at least 90% sequence identity to any one of SEQ IDNOs: 1827-3637.

In some aspects, the present disclosure provides for an engineerednuclease system comprising: (a) an endonuclease comprising a RuvC_IIIdomain having at least 75% sequence identity to any one of SEQ ID NOs:1827-3637; and (b) an engineered guide ribonucleic acid structureconfigured to form a complex with the endonuclease comprising: (i) aguide ribonucleic acid sequence configured to hybridize to a targetdeoxyribonucleic acid sequence; and (ii) a tracr ribonucleic acidsequence configured to bind to the endonuclease.

In some aspects, the present disclosure provides for an engineerednuclease system comprising: (a) an endonuclease configured to bind to aprotospacer adjacent motif (PAM) sequence comprising SEQ ID NOs:5512-5537, wherein the endonuclease is a class 2, type II Casendonuclease; and (b) an engineered guide ribonucleic acid structureconfigured to form a complex with the endonuclease comprising: (i) aguide ribonucleic acid sequence configured to hybridize to a targetdeoxyribonucleic acid sequence; and (ii) a tracr ribonucleic acidsequence configured to bind to the endonuclease.

In some embodiments, the endonuclease is derived from an uncultivatedmicroorganism. In some embodiments, the endonuclease has not beenengineered to bind to a different PAM sequence. In some embodiments, theendonuclease is not a Cas9 endonuclease, a Cas14 endonuclease, a Cas12aendonuclease, a Cas12b endonuclease, a Cas 12c endonuclease, a Cas12dendonuclease, a Cas12e endonuclease, a Cas13a endonuclease, a Cas13bendonuclease, a Cas13c endonuclease, or a Cas 13d endonuclease. In someembodiments, the endonuclease has less than 80% identity to a Cas9endonuclease. In some embodiments, the endonuclease further comprises anHNH domain. In some embodiments, the tracr ribonucleic acid sequencecomprises a sequence with at least 80% sequence identity to about 60 to90 consecutive nucleotides selected from any one of SEQ ID NOs:5476-5511 and SEQ ID NO: 5538.

In some aspects, the present disclosure provides for an engineerednuclease system comprising, (a) an engineered guide ribonucleic acidstructure comprising: (i) a guide ribonucleic acid sequence configuredto hybridize to a target deoxyribonucleic acid sequence; and (ii) atracr ribonucleic acid sequence configured to bind to an endonuclease,wherein the tracr ribonucleic acid sequence comprises a sequence with atleast 80% sequence identity to about 60 to 90 consecutive nucleotidesselected from any one of SEQ ID NOs: 5476-5511 and SEQ ID NO: 5538; and(b) a class 2, type II Cas endonuclease configured to bind to theengineered guide ribonucleic acid. In some embodiments, the endonucleaseis configured to bind to a protospacer adjacent motif (PAM) sequenceselected from the group comprising SEQ ID NOs: 5512-5537.

In some embodiments, the engineered guide ribonucleic acid structurecomprises at least two ribonucleic acid polynucleotides. In someembodiments, the engineered guide ribonucleic acid structure comprisesone ribonucleic acid polynucleotide comprising the guide ribonucleicacid sequence and the tracr ribonucleic acid sequence.

In some embodiments, the guide ribonucleic acid sequence iscomplementary to a prokaryotic, bacterial, archaeal, eukaryotic, fungal,plant, mammalian, or human genomic sequence. In some embodiments, theguide ribonucleic acid sequence is 15-24 nucleotides in length. In someembodiments, the endonuclease comprises one or more nuclear localizationsequences (NLSs) proximal to an N- or C-terminus of the endonuclease. Insome embodiments, the NLS comprises a sequence selected from SEQ ID NOs:5597-5612.

In some embodiments, the engineered nuclease system further comprises asingle- or double-stranded DNA repair template comprising from 5′ to 3′:a first homology arm comprising a sequence of at least 20 nucleotides 5′to the target deoxyribonucleic acid sequence, a synthetic DNA sequenceof at least 10 nucleotides, and a second homology arm comprising asequence of at least 20 nucleotides 3′ to the target sequence. In someembodiments, the first or second homology arm comprises a sequence of atleast 40, 80, 120, 150, 200, 300, 500, or 1,000 nucleotides.

In some embodiments, the system further comprises a source of Mg²⁺

In some embodiments, the endonuclease and the tracr ribonucleic acidsequence are derived from distinct bacterial species within a samephylum. In some embodiments, the endonuclease is derived from abacterium belonging to a genus Dermabacter. In some embodiments, theendonuclease is derived from a bacterium belonging to PhylumVerrucomicrobia, Phylum Candidatus Peregrinibacteria, or PhylumCandidatus Melainabacteria. In some embodiments, the endonuclease isderived from a bacterium comprising a 16S rRNA gene having at least 90%identity to any one of SEQ ID NOs: 5592-5595.

In some embodiments, the HNH domain comprises a sequence with at least70% or at least 80% identity to any one of SEQ ID NOs: 5638-5460. Insome embodiments, the endonuclease comprises SEQ ID NOs: 1-1826 or avariant thereof having at least 55% identity thereto. In someembodiments, the endonuclease comprises a sequence at least 70%, 80%, or90% identical to a sequence selected from the group consisting of SEQ IDNOs: 1827-1830 or SEQ ID NOs: 1827-2140.

In some embodiments, the endonuclease comprises a sequence at least 70%,80%, or 90% identical to a sequence selected from the group consistingof SEQ ID NOs: 3638-3641 or SEQ ID NOs: 3638-3954. In some embodiments,the endonuclease comprises at least 1, at least 2, at least 3, at least4, or at least 5 peptide motifs selected from the group consisting ofSEQ ID NOs: 5615-5632. In some embodiments, the endonuclease comprises asequence at least 70%, 80%, or 90% identical to a sequence selected fromthe group consisting of SEQ ID NOs: 1-4 or SEQ ID NOs: 1-319.

In some embodiments, the guide RNA structure comprises a sequence atleast 70%, 80%, or 90% identical to a sequence selected from the groupconsisting of SEQ ID NOs: 5461-5464, SEQ ID NOs: 5476-5479, or SEQ IDNOs: 5476-5489. In some embodiments, the guide RNA structure comprisesan RNA sequence predicted to comprise a hairpin consisting of a stem anda loop, wherein the stem comprises at least 10, at least 12 or at least14 base-paired ribonucleotides, and an asymmetric bulge within 4 basepairs of the loop.

In some embodiments, the endonuclease is configured to bind to a PAMcomprising a sequence selected from the group consisting of SEQ ID NOs:5512-5515 or SEQ ID NOs: 5527-5530.

In some embodiments: (a) the endonuclease comprises a sequence at least70%, at least 80%, or at least 90% identical to SEQ ID NO: 1827; (b) theguide RNA structure comprises a sequence at least 70%, at least 80%, orat least 90% identical to at least one of SEQ ID NO: 5461 or SEQ ID NO:5476; and (c) the endonuclease is configured to bind to a PAM comprisingSEQ ID NO: 5512 or SEQ ID NO: 5527. In some embodiments: (a) theendonuclease comprises a sequence at least 70%, at least 80%, or atleast 90% identical to SEQ ID NO: 1828; (b) the guide RNA structurecomprises a sequence at least 70%, at least 80%, or at least 90%identical to at least one of SEQ ID NO: 5462 or SEQ ID NO: 5477; and (c)the endonuclease is configured to bind to a PAM comprising SEQ ID NO:5513 or SEQ ID NO: 5528. In some embodiments: (a) the endonucleasecomprises a sequence at least 70%, at least 80%, or at least 90%identical to SEQ ID NO: 1829; (b) the guide RNA structure comprises asequence at least 70%, at least 80%, or at least 90% identical to atleast one of SEQ ID NO: 5463 or SEQ ID NO: 5478; and (c) theendonuclease is configured to bind to a PAM comprising SEQ ID NO: 5514or SEQ ID NO: 5529. In some embodiments: (a) the endonuclease comprisesa sequence at least 70%, at least 80%, or at least 90% identical to SEQID NO: 1830; (b) the guide RNA structure comprises a sequence at least70%, at least 80%, or at least 90% identical to at least one of SEQ IDNO: 5464 or SEQ ID NO: 5479; and (c) the endonuclease is configured tobind to a PAM comprising SEQ ID NO: 5515 or SEQ ID NO: 5530.

In some embodiments, the endonuclease comprises a sequence at least 70%,80%, or 90% identical to a sequence selected from the group consistingof SEQ ID NOs: 2141-2142 or SEQ ID NOs: 2141-2241. In some embodiments,the endonuclease comprises a sequence at least 70%, 80%, or 90%identical to a sequence selected from the group consisting of SEQ IDNOs: 3955-3956 or SEQ ID NOs: 3955-4055. In some embodiments, theendonuclease comprises at least 1, at least 2, at least 3, at least 4,or at least 5 peptide motifs selected from the group consisting of SEQID NOs: 5632-5638. In some embodiments, the endonuclease comprises asequence at least 70%, 80%, or 90% identical to a sequence selected fromthe group consisting of SEQ ID NOs: 320-321 or SEQ ID NOs: 320-420. Insome embodiments, the guide RNA structure comprises a sequence at least70%, 80%, or 90% identical to a sequence selected from the groupconsisting of SEQ ID NO: 5465, SEQ ID NOs: 5490-5491 or SEQ ID NOs:5490-5494. In some embodiments, the guide RNA structure comprises atracr ribonucleic acid sequence comprising a hairpin comprising at least8, at least 10, or at least 12 base-paired ribonucleotides. In someembodiments, the endonuclease is configured to bind to a PAM comprisinga sequence selected from the group consisting of SEQ ID NOs: 5516 andSEQ ID NOs: 5531. In some embodiments: (a) the endonuclease comprises asequence at least 70%, 80%, or 90% identical to SEQ ID NO: 2141; (b) theguide RNA structure comprises a sequence at least 70%, 80%, or 90%identical to SEQ ID NO: 5490; and (c) the endonuclease is configured tobinding to a PAM comprising SEQ ID NO: 5531. In some embodiments: (a)the endonuclease comprises a sequence at least 70%, 80%, or 90%identical to SEQ ID NO: 2142; (b) the guide RNA structure comprises asequence at least 70%, 80%, or 90% identical to SEQ ID NO: 5465 or SEQID NO: 5491; and (c) the endonuclease is configured to binding to a PAMcomprising SEQ ID NO: 5516.

In some embodiments, the endonuclease comprises a sequence at least 70%,80%, or 90% identical to a sequence selected from the group consistingof SEQ ID NOs: 2245-2246. In some embodiments, the endonucleasecomprises a sequence at least 70%, 80%, or 90% identical to a sequenceselected from the group consisting of SEQ ID NOs: 4059-4060. In someembodiments, the endonuclease comprises at least 1, at least 2, at least3, at least 4, or at least 5 peptide motifs selected from the groupconsisting of SEQ ID NOs: 5639-5648. In some embodiments, theendonuclease comprises a sequence at least 70%, 80%, or 90% identical toa sequence selected from the group consisting of SEQ ID NOs: 424-425. Insome embodiments, the guide RNA structure comprises a sequence at least70%, 80%, or 90% identical to a sequence selected from the groupconsisting of SEQ ID NOs: 5498-5499 and SEQ ID NO: 5539. In someembodiments, the guide RNA structure comprises a guide ribonucleic acidsequence predicted to comprise a hairpin with an uninterruptedbase-paired region comprising at least 8 nucleotides of a guideribonucleic acid sequence and at least 8 nucleotides of a tracrribonucleic acid sequence, and wherein the tracr ribonucleic acidsequence comprises, from 5′ to 3′, a first hairpin and a second hairpin,wherein the first hairpin has a longer stem than the second hairpin.

In some embodiments, the endonuclease comprises a sequence at least 70%,80%, or 90% identical to a sequence selected from the group consistingof SEQ ID NOs: 2242-2244 or SEQ ID NOs: 2247-2249. In some embodiments,the endonuclease comprises a sequence at least 70%, 80%, or 90%identical to a sequence selected from the group consisting of SEQ IDNOs: 4056-4058 and SEQ ID NOs 4061-4063. In some embodiments, theendonuclease comprises at least 1, at least 2, at least 3, at least 4,or at least 5 peptide motifs selected from the group consisting of SEQID NOs: 5639-5648. In some embodiments, the endonuclease comprises asequence at least 70%, 80%, or 90% identical to a sequence selected fromthe group consisting of SEQ ID NOs: 421-423 or SEQ ID NOs: 426-428. Insome embodiments, the guide RNA structure comprises a sequence at least70%, 80%, or 90% identical to a sequence selected from the groupconsisting of SEQ ID NOs: 5466-5467, SEQ ID NOs: 5495-5497, SEQ ID NO:5500-5502, and SEQ ID NO: 5539. In some embodiments, the guide RNAstructure comprises a guide ribonucleic acid sequence predicted tocomprise a hairpin with an uninterrupted base-paired region comprisingat least 8 nucleotides of a guide ribonucleic acid sequence and at least8 nucleotides of a tracr ribonucleic acid sequence, and wherein thetracr ribonucleic acid sequence comprises, from 5′ to 3′, a firsthairpin and a second hairpin, wherein the first hairpin has a longerstem than the second hairpin. In some embodiments, the endonuclease isconfigured to binding to a PAM comprising a sequence selected from thegroup consisting of SEQ ID NOs: 5517-5518 or SEQ ID NOs: 5532-5534. Insome embodiments: (a) the endonuclease comprises a sequence at least70%, 80%, or 90% identical to SEQ ID NO: 2247; (b) the guide RNAstructure comprises a sequence at least 70%, 80%, or 90% identical toSEQ ID NO: 5500; and (c) the endonuclease is configured to binding to aPAM comprising SEQ ID NO: 5517 or SEQ ID NO: 5532. In some embodiments:(a) the endonuclease comprises a sequence at least 70%, 80%, or 90%identical to SEQ ID NO: 2248; (b) the guide RNA structure comprises asequence at least 70%, 80%, or 90% identical to SEQ ID NO: 5501; and (c)the endonuclease is configured to binding to a PAM comprising SEQ ID NO:5518 or SEQ ID NOs: 5533. In some embodiments: (a) the endonucleasecomprises a sequence at least 70%, 80%, or 90% identical to SEQ ID NO:2249; (b) the guide RNA structure comprises a sequence at least 70%,80%, or 90% identical to SEQ ID NO: 5502; and (c) the endonuclease isconfigured to binding to a PAM comprising SEQ ID NO: 5534.

In some embodiments, the endonuclease comprises a sequence at least 70%,80%, or 90% identical to a sequence selected from the group consistingof SEQ ID NO: 2253 or SEQ ID NOs: 2253-2481. In some embodiments, theendonuclease comprises a sequence at least 70%, 80%, or 90% identical toa sequence selected from the group consisting of SEQ ID NO: 4067 or SEQID NOs: 4067-4295. In some embodiments, the endonuclease comprises apeptide motif according to SEQ ID NO: 5649. In some embodiments, theendonuclease comprises a sequence at least 70%, 80%, or 90% identical toa sequence selected from the group consisting of SEQ ID NO: 432 or SEQID NOs: 432-660. In some embodiments, the guide RNA structure comprisesa sequence at least 70%, 80%, or 90% identical to a sequence selectedfrom the group consisting of SEQ ID NO: 5468 or SEQ ID NO: 5503. In someembodiments, the endonuclease is configured to binding to a PAMcomprising a sequence selected from the group consisting of SEQ ID NOs:5519. In some embodiments: (a) the endonuclease comprises a sequence atleast 70%, 80%, or 90% identical to SEQ ID NO: 2253; (b) the guide RNAstructure comprises a sequence at least 70%, 80%, or 90% identical toSEQ ID NO: 5468 or SEQ ID NO: 5503; and (c) the endonuclease isconfigured to binding to a PAM comprising SEQ ID NO: 5519.

In some embodiments, the endonuclease comprises a sequence at least 70%,80%, or 90% identical to a sequence selected from the group consistingof SEQ ID NOs: 2482-2489. In some embodiments, the endonucleasecomprises a sequence at least 70%, 80%, or 90% identical to a sequenceselected from the group consisting of SEQ ID NOs: 4296-4303. In someembodiments, the endonuclease comprises a sequence at least 70%, 80%, or90% identical to a sequence selected from the group consisting of or SEQID NOs: 661-668. In some embodiments, the endonuclease comprises asequence at least 70%, 80%, or 90% identical to a sequence selected fromthe group consisting of or SEQ ID NOs: 2490-2498. In some embodiments,the endonuclease comprises a sequence at least 70%, 80%, or 90%identical to a sequence selected from the group consisting of SEQ IDNOs: 4304-4312. In some embodiments, the endonuclease comprises asequence at least 70%, 80%, or 90% identical to a sequence selected fromthe group consisting of SEQ ID NOs: 669-677. In some embodiments, theguide RNA structure comprises a sequence at least 70%, 80%, or 90%identical to a sequence selected from the group consisting of SEQ ID NO:5504.

In some embodiments, the endonuclease comprises a sequence at least 70%,80%, or 90% identical to a sequence selected from the group consistingof SEQ ID NO: 2499 or SEQ ID NOs: 2499-2750. In some embodiments, theendonuclease comprises a sequence at least 70%, 80%, or 90% identical toa sequence selected from the group consisting of SEQ ID NO: 4313 or SEQID NOs: 4313-4564. In some embodiments, the endonuclease comprises atleast 1, at least 2, at least 3, at least 4, or at least 5 peptidemotifs selected from the group consisting of SEQ ID NOs: 5650-5667. Insome embodiments, the endonuclease comprises a sequence at least 70%,80%, or 90% identical to a sequence selected from the group consistingof SEQ ID NO: 678 or SEQ ID NOs: 678-929. In some embodiments, the guideRNA structure comprises a sequence at least 70%, 80%, or 90% identicalto SEQ ID NO: 5469 or SEQ ID NO: 5505. In some embodiments, theendonuclease is configured to binding to a PAM comprising SEQ ID NOs:5520 or SEQ ID NOs: 5535. In some embodiments: (a) the endonucleasecomprises a sequence at least 70%, 80%, or 90% identical to SEQ ID NO:2499; (b) the guide RNA structure comprises a sequence at least 70%,80%, or 90% identical to SEQ ID NO: 5469 or SEQ ID NO: 5505; and (c) theendonuclease is configured to binding to a PAM comprising SEQ ID NO:5520 or SEQ ID NO: 5535.

In some embodiments, the endonuclease comprises a sequence at least 70%,80%, or 90% identical to a sequence selected from the group consistingof SEQ ID NO: 2751 or SEQ ID NOs: 2751-2913. In some embodiments, theendonuclease comprises a sequence at least 70%, 80%, or 90% identical toa sequence selected from the group consisting of SEQ ID NO: 4565 or SEQID NOs: 4565-4727. In some embodiments, the endonuclease comprises atleast 1, at least 2, at least 3, at least 4, or at least 5 peptidemotifs selected from the group consisting of SEQ ID NOs: 5668-5678. Insome embodiments, the endonuclease comprises a sequence at least 70%,80%, or 90% identical to a sequence selected from the group consistingof SEQ ID NO: 930 or SEQ ID NOs: 930-1092. In some embodiments, theguide RNA structure comprises a sequence at least 70%, 80%, or 90%identical to SEQ ID NO: 5470 or SEQ ID NOs: 5506. In some embodiments,the endonuclease is configured to binding to a PAM comprising a sequenceselected from the group consisting of SEQ ID NOs: 5521 or SEQ ID NOs:5536. In some embodiments: (a) the endonuclease comprises a sequence atleast 70%, 80%, or 90% identical to SEQ ID NO: 2751; (b) the guide RNAstructure comprises a sequence at least 70%, 80%, or 90% identical toSEQ ID NO: 5470 or SEQ ID NO: 5506; and (c) the endonuclease isconfigured to binding to a PAM comprising SEQ ID NO: 5521 or SEQ ID NO:5536.

In some embodiments, the endonuclease comprises a sequence at least 70%,80%, or 90% identical to a sequence selected from the group consistingof SEQ ID NO: 2914 or SEQ ID NOs: 2914-3174. In some embodiments, theendonuclease comprises a sequence at least 70%, 80%, or 90% identical toa sequence selected from the group consisting of SEQ ID NO: 4728 or SEQID NOs: 4728-4988. In some embodiments, the endonuclease comprises atleast 1, at least 2, or at least 3 peptide motifs selected from thegroup consisting of SEQ ID NOs: 5676-5678. In some embodiments, theendonuclease comprises a sequence at least 70%, 80%, or 90% identical toa sequence selected from the group consisting of SEQ ID NO: 1093 or SEQID NOs: 1093-1353. In some embodiments, the guide RNA structurecomprises a sequence at least 70%, 80%, or 90% identical to a sequenceselected from the group consisting of SEQ ID NO: 5471, SEQ ID NO: 5507,and SEQ ID NOs: 5540-5542. In some embodiments, the guide RNA structurecomprises a tracr ribonucleic acid sequence predicted to comprise atleast two hairpins comprising less than 5 base-paired ribonucleotides.In some embodiments, the endonuclease is configured to binding to a PAMcomprising SEQ ID NO: 5522. In some embodiments: (a) the endonucleasecomprises a sequence at least 70%, 80%, or 90% identical to SEQ ID NO:2914; (b) the guide RNA structure comprises a sequence at least 70%,80%, or 90% identical to SEQ ID NO: 5471 or SEQ ID NO: 5507; and (c) theendonuclease is configured to binding to a PAM comprising SEQ ID NO:5522.

In some embodiments, the endonuclease comprises a sequence at least 70%,80%, or 90% identical to a sequence selected from the group consistingof SEQ ID NO: 3175 or SEQ ID NOs: 3175-3330. In some embodiments, theendonuclease comprises a sequence at least 70%, 80%, or 90% identical toa sequence selected from the group consisting of SEQ ID NO: 4989 or SEQID NOs: 4989-5146. In some embodiments, the endonuclease comprises atleast 1, at least 2, at least 3, at least 4, or at least 5 peptidemotifs selected from the group consisting of SEQ ID NOs: 5679-5686. Insome embodiments, the endonuclease comprises a sequence at least 70%,80%, or 90% identical to a sequence selected from the group consistingof SEQ ID NO: 1354 or SEQ ID NOs: 1354-1511. In some embodiments, theguide RNA structure comprises a sequence at least 70%, 80%, or 90%identical to a sequence selected from the group consisting of SEQ IDNOs: 5472 or SEQ ID NOs: 5508. In some embodiments, the endonuclease isconfigured to binding to a PAM comprising a sequence selected from thegroup consisting of SEQ ID NO: 5523 or SEQ ID NO: 5537. In someembodiments: (a) the endonuclease comprises a sequence at least 70%,80%, or 90% identical to SEQ ID NO: 3175; (b) the guide RNA structurecomprises a sequence at least 70%, 80%, or 90% identical to SEQ ID NO:5472 or SEQ ID NO: 5508; and (c) the endonuclease is configured tobinding to a PAM comprising SEQ ID NO: 5523 or SEQ ID NO: 5537.

In some embodiments, the endonuclease comprises a sequence at least 70%,80%, or 90% identical to a sequence selected from the group consistingof SEQ ID NOs: 3331 or SEQ ID NOs: 3331-3474. In some embodiments, theendonuclease comprises a sequence at least 70%, 80%, or 90% identical toa sequence selected from the group consisting of SEQ ID NOs: 5147 or SEQID NOs: 5147-5290. In some embodiments, the endonuclease comprises atleast 1, at least 2, at least 3, at least 4, or at least 5 peptidemotifs selected from the group consisting of SEQ ID NOs: 5674-5675 andSEQ ID NOs: 5687-5693. In some embodiments, the endonuclease comprises asequence at least 70%, 80%, or 90% identical to a sequence selected fromthe group consisting of SEQ ID NO: 1512 or SEQ ID NOs: 1512-1655. Insome embodiments, the guide RNA structure comprises a sequence at least70%, 80%, or 90% identical to a sequence selected from the groupconsisting of SEQ ID NO: 5473 or SEQ ID NO: 5509. In some embodiments,the endonuclease is configured to binding to a PAM comprising SEQ ID NO:5524. In some embodiments: (a) the endonuclease comprises a sequence atleast 70%, 80%, or 90% identical to SEQ ID NO: 3331; (b) the guide RNAstructure comprises a sequence at least 70%, 80%, or 90% identical toSEQ ID NO: 5473 or SEQ ID NO: 5509; and (c) the endonuclease isconfigured to binding to a PAM comprising SEQ ID NO: 5524.

In some embodiments, the endonuclease comprises a sequence at least 70%,80%, or 90% identical to a sequence selected from the group consistingof SEQ ID NO: 3475 or SEQ ID NOs: 3475-3568. In some embodiments, theendonuclease comprises a sequence at least 70%, 80%, or 90% identical toa sequence selected from the group consisting of SEQ ID NO: 5291 or SEQID NOs: 5291-5389. In some embodiments, the endonuclease comprises atleast 1, at least 2, at least 3, at least 4, or at least 5 peptidemotifs selected from the group consisting of SEQ ID NOs: 5694-5699. Insome embodiments, the endonuclease comprises a sequence at least 70%,80%, or 90% identical to a sequence selected from the group consistingof SEQ ID NO: 1656 or SEQ ID NOs: 1656-1755. In some embodiments, theguide RNA structure comprises a sequence at least 70%, 80%, or 90%identical to SEQ ID NO: 5474 or SEQ ID NO: 5510. In some embodiments,the endonuclease is configured to binding to a PAM comprising SEQ IDNOs: 5525. In some embodiments: (a) the endonuclease comprises asequence at least 70%, 80%, or 90% identical to SEQ ID NO: 3475; (b) theguide RNA structure comprises a sequence at least 70%, 80%, or 90%identical to SEQ ID NO: 5474 or SEQ ID NO: 5510; and (c) theendonuclease is configured to binding to a PAM comprising SEQ ID NO:5525.

In some embodiments, the endonuclease comprises a sequence at least 70%,80%, or 90% identical to a sequence selected from the group consistingof SEQ ID NO: 3569 or SEQ ID NOs: 3569-3637. In some embodiments, theendonuclease comprises a sequence at least 70%, 80%, or 90% identical toa sequence selected from the group consisting of SEQ ID NO: 5390 or SEQID NOs: 5390-5460. In some embodiments, the endonuclease comprises atleast 1, at least 2, at least 3, at least 4, or at least 5 peptidemotifs selected from the group consisting of SEQ ID NOs: 5700-5717. Insome embodiments, the endonuclease comprises a sequence at least 70%,80%, or 90% identical to a sequence selected from the group consistingof SEQ ID NO: 1756 or SEQ ID NOs: 1756-1826. In some embodiments, theguide RNA structure comprises a sequence at least 70%, 80%, or 90%identical to SEQ ID NO: 5475 or SEQ ID NOs: 5511. In some embodiments,the endonuclease is configured to binding to a PAM comprising SEQ ID NO:5526. In some embodiments: (a) the endonuclease comprises a sequence atleast 70%, 80%, or 90% identical to SEQ ID NO: 3569; (b) the guide RNAstructure comprises a sequence at least 70%, 80%, or 90% identical toSEQ ID NO: 5475 or SEQ ID NO: 5511; and (c) the endonuclease isconfigured to binding to a PAM comprising SEQ ID NO: 5526. In someembodiments, the sequence identity is determined by a BLASTP, CLUSTALW,MUSCLE, MAFFT, or Smith-Waterman homology search algorithm. In someembodiments, the sequence identity is determined by the BLASTP homologysearch algorithm using parameters of a wordlength (W) of 3, anexpectation (E) of 10, and a BLOSUM62 scoring matrix setting gap costsat existence of 11, extension of 1, and using a conditionalcompositional score matrix adjustment.

In some aspects, the present disclosure provides for an engineered guideribonucleic acid polynucleotide comprising: (a) a DNA-targeting segmentcomprising a nucleotide sequence that is complementary to a targetsequence in a target DNA molecule; and (b) a protein-binding segmentcomprising two complementary stretches of nucleotides that hybridize toform a double-stranded RNA (dsRNA) duplex, wherein the two complementarystretches of nucleotides are covalently linked to one another withintervening nucleotides, and wherein the engineered guide ribonucleicacid polynucleotide is configured to forming a complex with anendonuclease comprising a RuvC_III domain having at least 75% sequenceidentity to any one of SEQ ID NOs: 1827-3637 and targeting the complexto the target sequence of the target DNA molecule. In some embodiments,the DNA-targeting segment is positioned 5′ of both of the twocomplementary stretches of nucleotides.

In some embodiments: (a) the protein binding segment comprises asequence having at least 70%, at least 80%, or at least 90% identity toa sequence selected from the group consisting of SEQ ID NOs: 5476-5479or SEQ ID NOs: 5476-5489; (b) the protein binding segment comprises asequence having at least 70%, at least 80%, or at least 90% identity toa sequence selected from the group consisting of (SEQ ID NOs: 5490-5491or SEQ ID NOs: 5490-5494) and SEQ ID NO: 5538; (c) the protein bindingsegment comprises a sequence having at least 70%, at least 80%, or atleast 90% identity to a sequence selected from the group consisting ofSEQ ID NOs: 5498-5499; (d) the protein binding segment comprises asequence having at least 70%, at least 80%, or at least 90% identity toa sequence selected from the group consisting of SEQ ID NOs: 5495-5497and SEQ ID NOs: 5500-5502; (e) the protein binding segment comprises asequence having at least 70%, at least 80%, or at least 90% identity toSEQ ID NO: 5503; (f) the protein binding segment comprises a sequencehaving at least 70%, at least 80%, or at least 90% identity to SEQ IDNO: 5504; (g) the protein binding segment comprises a sequence having atleast 70%, at least 80%, or at least 90% identity to SEQ ID NOs: 5505;(h) protein binding segment comprises a sequence having at least 70%, atleast 80%, or at least 90% identity to SEQ ID NO: 5506; (i) proteinbinding segment comprises a sequence having at least 70%, at least 80%,or at least 90% identity to SEQ ID NO: 5507; (j) the protein bindingsegment comprises a sequence having at least 70%, at least 80%, or atleast 90% identity to SEQ ID NO: 5508; (k) the protein binding segmentcomprises a sequence having at least 70%, at least 80%, or at least 90%identity to SEQ ID NO: 5509; (1) the protein binding segment comprises asequence having at least 70%, at least 80%, or at least 90% identity toSEQ ID NO: 5510; or (m) the protein binding segment comprises a sequencehaving at least 70%, at least 80%, or at least 90% identity to SEQ IDNO: 5511.

In some embodiments: (a) the guide ribonucleic acid polynucleotidecomprises an RNA sequence comprising a hairpin comprising a stem and aloop, wherein the stem comprises at least 10, at least 12, or at least14 base-paired ribonucleotides, and an asymmetric bulge within 4 basepairs of the loop; (b) the guide ribonucleic acid polynucleotidecomprises a tracr ribonucleic acid sequence predicted to comprise ahairpin comprising at least 8, at least 10, or at least 12 base-pairedribonucleotides; (c) the guide ribonucleic acid polynucleotide comprisesa guide ribonucleic acid sequence predicted to comprise a hairpin withan uninterrupted base-paired region comprising at least 8 nucleotides ofa guide ribonucleic acid sequence and at least 8 nucleotides of a tracrribonucleic acid sequence, and wherein the tracr ribonucleic acidsequence comprises, from 5′ to 3′, a first hairpin and a second hairpin,wherein the first hairpin has a longer stem than the second hairpin; or(d) the guide ribonucleic acid polynucleotide comprises a tracrribonucleic acid sequence predicted to comprise at least two hairpinscomprising less than 5 base-paired ribonucleotides.

In some aspects, the present disclosure provides for a deoxyribonucleicacid polynucleotide encoding any of the engineered guide ribonucleicacid polynucleotides described herein.

In some aspects, the present disclosure provides for a nucleic acidcomprising an engineered nucleic acid sequence optimized for expressionin an organism, wherein the nucleic acid encodes a class 2, type II Casendonuclease comprising a RuvC_III domain and an HNH domain, and whereinthe endonuclease is derived from an uncultivated microorganism.

In some aspects, the present disclosure provides for a nucleic acidcomprising an engineered nucleic acid sequence optimized for expressionin an organism, wherein the nucleic acid encodes an endonucleasecomprising a RuvC_III domain having at least 70% sequence identity toany one of SEQ ID NOs: 1827-3637. In some embodiments, the endonucleasecomprises an HNH domain having at least 70% or at least 80% sequenceidentity to any one of SEQ ID NOs: 3638-5460. In some embodiments, theendonuclease comprises SEQ ID NOs: 5572-5591 or a variant thereof havingat least 70% sequence identity thereto. In some embodiments, theendonuclease comprises a sequence encoding one or more nuclearlocalization sequences (NLSs) proximal to an N- or C-terminus of theendonuclease. In some embodiments, the NLS comprises a sequence selectedfrom SEQ ID NOs: 5597-5612.

In some embodiments, the organism is prokaryotic, bacterial, eukaryotic,fungal, plant, mammalian, rodent, or human. In some embodiments, theorganism is E. coli, and: (a) the nucleic acid sequence has at least70%, 80%, or 90% identity to a sequence selected from the groupconsisting of SEQ ID NOs: 5572-5575; (b) the nucleic acid sequence hasat least 70%, 80%, or 90% identity to a sequence selected from the groupconsisting of SEQ ID NOs: 5576-5577; (c) the nucleic acid sequence hasat least 70%, 80%, or 90% identity to a sequence selected from the groupconsisting of SEQ ID NOs: 5578-5580; (d) the nucleic acid sequence hasat least 70%, 80%, or 90% identity to SEQ ID NO: 5581; (e) the nucleicacid sequence has at least 70%, 80%, or 90% identity to SEQ ID NO: 5582;(f) the nucleic acid sequence has at least 70%, 80%, or 90% identity toSEQ ID NO: 5583; (g) the nucleic acid sequence has at least 70%, 80%, or90% identity to SEQ ID NO: 5584; (h) the nucleic acid sequence has atleast 70%, 80%, or 90% identity to SEQ ID NO: 5585; (i) the nucleic acidsequence has at least 70%, 80%, or 90% identity to SEQ ID NO: 5586; or(j) the nucleic acid sequence has at least 70%, 80%, or 90% identity toSEQ ID NO: 5587. In some embodiments, the organism is human, and: (a)the nucleic acid sequence has at least 70%, 80%, or 90% identity to SEQID NO: 5588 or SEQ ID NO: 5589; or (b) the nucleic acid sequence has atleast 70%, 80%, or 90% identity to SEQ ID NO: 5590 or SEQ ID NO: 5591.

In some aspects, the present disclosure provides for a vector comprisinga nucleic acid sequence encoding a class 2, type II Cas endonucleasecomprising a RuvC_III domain and an HNH domain, wherein the endonucleaseis derived from an uncultivated microorganism.

In some aspects, the present disclosure provides for a vector comprisingthe any of the nucleic acids described herein. In some embodiments, thevector further comprises a nucleic acid encoding an engineered guideribonucleic acid structure configured to form a complex with theendonuclease comprising: (a) a guide ribonucleic acid sequenceconfigured to hybridize to a target deoxyribonucleic acid sequence; and(b) a tracr ribonucleic acid sequence configured to binding to theendonuclease. In some embodiments, the vector is a plasmid, aminicircle, a CELiD, an adeno-associated virus (AAV) derived virion, ora lentivirus.

In some aspects, the present disclosure provides for a cell comprisingany of the vectors described herein.

In some aspects, the present disclosure provides for a method ofmanufacturing an endonuclease, comprising cultivating any of the cellsdescribed herein.

In some aspects, the present disclosure provides for a method forbinding, cleaving, marking, or modifying a double-strandeddeoxyribonucleic acid polynucleotide, comprising: (a) contacting thedouble-stranded deoxyribonucleic acid polynucleotide with a class 2,type II Cas endonuclease in complex with an engineered guide ribonucleicacid structure configured to bind to the endonuclease and thedouble-stranded deoxyribonucleic acid polynucleotide; (b) wherein thedouble-stranded deoxyribonucleic acid polynucleotide comprises aprotospacer adjacent motif (PAM); and (c) wherein the PAM comprises asequence selected from the group consisting of SEQ ID NOs: 5512-5526 orSEQ ID NOs: 5527-5537. In some embodiments, the double-strandeddeoxyribonucleic acid polynucleotide comprises a first strand comprisinga sequence complementary to a sequence of the engineered guideribonucleic acid structure and a second strand comprising the PAM. Insome embodiments, the PAM is directly adjacent to the 3′ end of thesequence complementary to the sequence of the engineered guideribonucleic acid structure.

In some embodiments, the class 2, type II Cas endonuclease is not a Cas9endonuclease, a Cas14 endonuclease, a Cas12a endonuclease, a Cas12bendonuclease, a Cas 12c endonuclease, a Cas12d endonuclease, a Cas12eendonuclease, a Cas13a endonuclease, a Cas13b endonuclease, a Cas13cendonuclease, or a Cas 13d endonuclease. In some embodiments, the class2, type II Cas endonuclease is derived from an uncultivatedmicroorganism. In some embodiments, the double-stranded deoxyribonucleicacid polynucleotide is a eukaryotic, plant, fungal, mammalian, rodent,or human double-stranded deoxyribonucleic acid polynucleotide.

In some embodiments: (a) the PAM comprises a sequence selected from thegroup consisting of SEQ ID NOs: 5512-5515 and SEQ ID NOs: 5527-5530; (b)the PAM comprises SEQ ID NO: 5516 or SEQ ID NO: 5531; (c) the PAMcomprises SEQ ID NO: 5539; (d) the PAM comprises SEQ ID NO: 5517 or SEQID NO: 5518; (e) the PAM comprises SEQ ID NO: 5519; (f) the PAMcomprises SEQ ID NO: 5520 or SEQ ID NO: 5535; (g) the PAM comprises SEQID NO: 5521 or SEQ ID NO: 5536; (h) the PAM comprises SEQ ID NO: 5522;(i) the PAM comprises SEQ ID NO: 5523 or SEQ ID NO: 5537; (j) the PAMcomprises SEQ ID NO: 5524; (k) the PAM comprises SEQ ID NO: 5525; or (1)the PAM comprises SEQ ID NO: 5526.

In some aspects, the present disclosure provides for a method ofmodifying a target nucleic acid locus, the method comprising deliveringto the target nucleic acid locus any of the engineered nuclease systemsdescribed herein, wherein the endonuclease is configured to form acomplex with the engineered guide ribonucleic acid structure, andwherein the complex is configured such that upon binding of the complexto the target nucleic acid locus, the complex modifies the targetnucleic locus. In some embodiments, modifying the target nucleic acidlocus comprises binding, nicking, cleaving, or marking the targetnucleic acid locus. In some embodiments, the target nucleic acid locuscomprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). In someembodiments, the target nucleic acid comprises genomic DNA, viral DNA,viral RNA, or bacterial DNA. In some embodiments, the target nucleicacid locus is in vitro. In some embodiments, the target nucleic acidlocus is within a cell. In some embodiments, the cell is a prokaryoticcell, a bacterial cell, a eukaryotic cell, a fungal cell, a plant cell,an animal cell, a mammalian cell, a rodent cell, a primate cell, or ahuman cell.

In some embodiments, delivering the engineered nuclease system to thetarget nucleic acid locus comprises delivering nucleic acid comprising:(i) an engineered nucleic acid sequence optimized for expression in anorganism, wherein said nucleic acid encodes a class 2, type II Casendonuclease comprising a RuvC III domain and an HNH domain, and whereinsaid endonuclease is derived from an uncultivated microorganism or (ii)an engineered nucleic acid sequence optimized for expression in anorganism, wherein said nucleic acid encodes an endonuclease comprising aRuvC III domain having at least 70% sequence identity to any one of SEQID NOs: 1827-3637, wherein the endonuclease optionally: (a) comprises anHNH domain having at least 70% or at least 80% sequence identity to anyone of SEQ ID NOs: 3638-5460; (b) comprises SEQ ID NOs: 5572-5591 or avariant thereof having at least 70% sequence identity thereto; (c)comprises a sequence encoding one or more nuclear localization sequences(NLSs) proximal to an N- or C-terminus of said endonuclease; or (d)comprises a sequence encoding one or more nuclear localization sequences(NLSs) proximal to an N- or C-terminus of said endonuclease wherein saidNLS comprises a sequence selected from SEQ ID NOs: 5597-5612. In someembodiments, delivering the engineered nuclease system to the targetnucleic acid locus comprises delivering a nucleic acid comprising anopen reading frame encoding the endonuclease. In some embodiments, thenucleic acid comprises a promoter to which the open reading frameencoding the endonuclease is operably linked. In some embodiments, theengineered nuclease system to the target nucleic acid locus comprisesdelivering a capped mRNA containing the open reading frame encoding theendonuclease. In some embodiments, the engineered nuclease system to thetarget nucleic acid locus comprises delivering a translated polypeptide.In some embodiments, the engineered nuclease system to the targetnucleic acid locus comprises delivering a deoxyribonucleic acid (DNA)encoding the engineered guide ribonucleic acid structure operably linkedto a ribonucleic acid (RNA) pol III promoter. In some embodiments, theendonuclease induces a single-stranded break or a double-stranded breakat or proximal to the target locus.

Additional aspects and advantages of the present disclosure will becomereadily apparent to those skilled in this art from the followingdetailed description, wherein only illustrative embodiments of thepresent disclosure are shown and described. As will be realized, thepresent disclosure is capable of other and different embodiments, andits several details are capable of modifications in various obviousrespects, all without departing from the disclosure. Accordingly, thedrawings and description are to be regarded as illustrative in nature,and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings (also “Figure” and “FIG.” herein), of which:

FIG. 1 depicts typical organizations of CRISPR/Cas loci of differentclasses and types.

FIG. 2 depicts the architecture of a natural Class2/Type IIcrRNA/tracrRNA pair, compared to a hybrid sgRNA wherein both are joined.

FIG. 3 depicts schematics showing organization of CRISPR loci encodingenzymes from the MG1 family.

FIG. 4 depicts schematics showing organization of CRISPR loci encodingenzymes from the MG2 family.

FIG. 5 depicts schematics showing organization of CRISPR loci encodingenzymes from the MG3 family.

FIG. 6A-6E depicts a structure-based alignment of an enzyme of thepresent disclosure (MG1-1) versus Cas9 from Staphylococcus aureus (SEQID NO:5613).

FIG. 7A-7D depicts a structure-based alignment of an enzyme of thepresent disclosure (MG2-1) versus Cas9 from Staphylococcus aureus (SEQID NO:5613).

FIG. 8A-8C depicts a structure-based alignment of an enzyme of thepresent disclosure (MG3-1) versus Cas9 from Actinomyces naeslundii (SEQID NO: 5614).

FIGS. 9A-90 depicts a structure-based alignment of MG1 family enzymesMG1-1 through MG1-6 (SEQ ID NOs: 5, 6, 9, 1, 2, and 3).

FIG. 10 depicts in vitro cleavage of DNA by MG1-4 in complex with itscorresponding sgRNA containing targeting sequences of varying lengths.

FIG. 11 depicts in cell cleavage of E. coli genomic DNA using MG1-4along with its corresponding sgRNA. Shown are dilution series of cellstransformed with MG1-4 along with target or non-target spacer (top);bottom panel shows the data quantitated, where the left bar representsnon-target sgRNA and the right bar represents target sgRNA.

FIG. 12 depicts in cell indel formation generated by transfection of HEKcells with MG1-4 or MG1-6 constructs described in Example 11 alongsidetheir corresponding sgRNAs containing various different targetingsequences targeting various locations in the human genome.

FIG. 13 depicts vitro cleavage of DNA by MG3-6 in complex with itscorresponding sgRNA containing targeting sequences of varying lengths.

FIG. 14 depicts in cell cleavage of E. coli genomic DNA using MG3-7along with its corresponding sgRNA. Shown are dilution series of cellstransformed with MG3-7 along with target or non-target spacer (top);bottom panel shows the data quantitated, where the left bar representsnon-target sgRNA and the right bar represents target sgRNA.

FIG. 15 depicts in cell indel formation generated by transfection of HEKcells with MG3-7 constructs described in Example 13 alongside theircorresponding sgRNAs containing various different targeting sequencestargeting various locations in the human genome.

FIG. 16 depicts in vitro cleavage of DNA by MG15-1 in complex with itscorresponding sgRNA containing targeting sequences of varying lengths.

FIGS. 17, 18, 19, and 20 depict agarose gels showing the results of PAMvector library cleavage in the presence of TXTL extracts containingvarious MG family nucleases and their corresponding tracrRNAs or sgRNAs.

FIGS. 21, 22, 23, 24, 25 and 26 depict predicted structures (predictede.g., as in Example 7) of corresponding sgRNAs of MG enzymes describedherein.

FIGS. 27, 28, 29, 30, 31, 32 and 33 depict seqLogo representations ofPAM sequences derived via NGS as described herein (e.g. as described inExample 6).

FIG. 34 depicts in cell cleavage of E. coli genomic DNA using MG2-7along with its corresponding sgRNA. Shown are dilution series of cellstransformed with MG2-7 along with target or non-target spacer (top);bottom panel shows the data quantitated, where the right bar representsnon-target sgRNA and the left bar represents target sgRNA.

FIG. 35 depicts in cell cleavage of E. coli genomic DNA using MG14-1along with its corresponding sgRNA. Shown are dilution series of cellstransformed with MG14-1 along with target or non-target spacer (top);bottom panel shows the data quantitated, where the right bar representsnon-target sgRNA and the left bar represents target sgRNA.

FIG. 36 depicts in cell cleavage of E. coli genomic DNA using MG15-1along with its corresponding sgRNA. Shown are dilution series of cellstransformed with MG15-1 along with target or non-target spacer (top);bottom panel shows the data quantitated, where the right bar representsnon-target sgRNA and the left bar represents target sgRNA.

FIGS. 37-39 depicts in cell indel formation generated by transfection ofHEK cells with MG1-4, MG1-6 and MG1-7 constructs described in Example 11alongside their corresponding sgRNAs containing various differenttargeting sequences targeting various locations in the human genome.

FIGS. 40-42 depicts in cell indel formation generated by transfection ofHEK cells with MG3-6, MG3-7 and MG3-8 constructs described in Example 13alongside their corresponding sgRNAs containing various differenttargeting sequences targeting various locations in the human genome.

FIG. 43 depicts in cell indel formation generated by transfection of HEKcells with MG14-1 constructs described in Example 14 alongside theircorresponding sgRNAs containing various different targeting sequencestargeting various locations in the human genome.

FIG. 44 depicts in cell indel formation generated by transfection of HEKcells with MG18-1 constructs described in Example 17 alongside theircorresponding sgRNAs containing various different targeting sequencestargeting various locations in the human genome.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

The Sequence Listing filed herewith provides exemplary polynucleotideand polypeptide sequences for use in methods, compositions and systemsaccording to the disclosure. Below are exemplary descriptions ofsequences therein.

MG1

SEQ ID NOs: 1-319 show the full-length peptide sequences of MG1nucleases.

SEQ ID NOs: 1827-2140 show the peptide sequences of RuvC_III domains ofMG1 nucleases above.

SEQ ID NOs: 3638-3955 show the peptide of HNH domains of MG1 nucleasesabove.

SEQ ID NOs: 5476-5479 show the nucleotide sequences of MG1 tracrRNAsderived from the same loci as MG1 nucleases above (e.g., same loci asSEQ ID NO:1-4, respectively).

SEQ ID NOs: 5461-5464 show the nucleotide sequences of sgRNAs engineeredto function with an MG1 nuclease (e.g., SEQ ID NO:1-4, respectively),where Ns denote nucleotides of a targeting sequence.

SEQ ID NOs: 5572-5575 show nucleotide sequences for E. colicodon-optimized coding sequences for MG1 family enzymes (SEQ ID NOs:1-4).

SEQ ID NOs: 5588-5589 show nucleotide sequences for humancodon-optimized coding sequences for MG1 family enzymes (SEQ ID NOs: 1and 3).

SEQ ID NOs: 5616-5632 show peptide motifs characteristic of MG1 familyenzymes.

MG2

SEQ ID NOs: 320-420 show the full-length peptide sequences of MG2nucleases.

SEQ ID NOs: 2141-2241 show the peptide sequences of RuvC_III domains ofMG2 nucleases above.

SEQ ID NOs: 3955-4055 show the peptide of HNH domains of MG2 nucleasesabove.

SEQ ID NOs: 5490-5494 show the nucleotide sequences of MG2 tracrRNAsderived from the same loci as MG2 nucleases above (e.g., same loci asSEQ ID NOs: 320, 321, 323, 325, and 326, respectively).

SEQ ID NO: 5465 shows the nucleotide sequence of an sgRNA engineered tofunction with an MG2 nuclease (e.g., SEQ ID NO: 321 above).

SEQ ID NOs: 5572-5575 show nucleotide sequences for E. colicodon-optimized coding sequences for MG2 family enzymes.

SEQ ID NOs: 5631-5638 show peptide sequences characteristic of MG2family enzymes.

MG3

SEQ ID NOs: 421-431 show the full-length peptide sequences of MG3nucleases.

SEQ ID NOs: 2242-2252 show the peptide sequences of RuvC_III domains ofMG3 nucleases above.

SEQ ID NOs: 4056-4066 show the peptide of HNH domains of MG3 nucleasesabove.

SEQ ID NOs: 5495-5502 show the nucleotide sequences of MG3 tracrRNAsderived from the same loci as MG3 nucleases above (e.g., same loci asSEQ ID NOs: 421-428, respectively).

SEQ ID NOs: 5466-5467 show the nucleotide sequence of sgRNAs engineeredto function with an MG3 nuclease (e.g., SEQ ID NOs: 421-423).

SEQ ID NOs: 5578-5580 show nucleotide sequences for E. colicodon-optimized coding sequences for MG3 family enzymes.

SEQ ID NOs: 5639-5648 show peptide sequences characteristic of MG3family enzymes.

MG4

SEQ ID NOs: 432-660 show the full-length peptide sequences of MG4nucleases.

SEQ ID NOs: 2253-2481 show the peptide sequences of RuvC_III domains ofMG4 nucleases above.

SEQ ID NOs: 4067-4295 show the peptide of HNH domains of MG4 nucleasesabove.

SEQ ID NO: 5503 shows the nucleotide sequences of an MG4 tracrRNAderived from the same loci as MG4 nucleases above.

SEQ ID NO: 5468 shows the nucleotide sequence of sgRNAs engineered tofunction with an MG4 nuclease.

SEQ ID NO: 5649 shows a peptide sequence characteristic of MG4 familyenzymes.

MG6

SEQ ID NOs: 661-668 show the full-length peptide sequences of MG6nucleases.

SEQ ID NOs: 2482-2489 show the peptide sequences of RuvC_III domains ofMG6 nucleases above.

SEQ ID NOs: 4296-4303 show the peptide of HNH domains of MG3 nucleasesabove.

MG7

SEQ ID NOs: 669-677 show the full-length peptide sequences of MG7nucleases.

SEQ ID NOs: 2490-2498 show the peptide sequences of RuvC_III domains ofMG7 nucleases above.

SEQ ID NOs: 4304-4312 show the peptide of HNH domains of MG3 nucleasesabove.

SEQ ID NO: 5504 shows the nucleotide sequence of an MG7 tracrRNA derivedfrom the same loci as MG7 nucleases above.

MG14

SEQ ID NOs: 678-929 show the full-length peptide sequences of MG14nucleases.

SEQ ID NOs: 2499-2750 show the peptide sequences of RuvC_III domains ofMG14 nucleases above.

SEQ ID NOs: 4313-4564 show the peptide of HNH domains of MG14 nucleasesabove.

SEQ ID NO: 5505 shows the nucleotide sequences of MG14 tracrRNA derivedfrom the same loci as MG14 nucleases above.

SEQ ID NO: 5581 shows a nucleotide sequence for an E. colicodon-optimized coding sequences for an MG14 family enzyme.

SEQ ID NOs: 5650-5667 show peptide sequences characteristic of MG14family enzymes.

MG15

SEQ ID NOs: 930-1092 show the full-length peptide sequences of MG15nucleases.

SEQ ID NOs: 2751-2913 show the peptide sequences of RuvC_III domains ofMG15 nucleases above.

SEQ ID NOs: 4565-4727 show the peptide of HNH domains of MG15 nucleasesabove.

SEQ ID NO: 5506 shows the nucleotide sequences of MG15 tracrRNA derivedfrom the same loci as MG15 nucleases above.

SEQ ID NOs: 5470 shows the nucleotide sequence of an sgRNA engineered tofunction with an MG15 nuclease.

SEQ ID NO: 5582 shows a nucleotide sequence for an E. colicodon-optimized coding sequences for an MG15 family enzyme.

SEQ ID NOs: 5668-5675 show peptide sequences characteristic of MG15family enzymes.

MG16

SEQ ID NOs: 1093-1353 show the full-length peptide sequences of MG16nucleases.

SEQ ID NOs: 2914-3174 show the peptide sequences of RuvC_III domains ofMG16 nucleases above.

SEQ ID NOs: 4728-4988 show the peptide of HNH domains of MG16 nucleasesabove.

SEQ ID NOs: 5507 show the nucleotide sequences of an MG16 tracrRNAderived from the same loci as MG3 nucleases above.

SEQ ID NOs: 5471 shows the nucleotide sequence of sgRNAs engineered tofunction with an MG16 nuclease.

SEQ ID NO: 5583 shows a nucleotide sequence for an E. colicodon-optimized coding sequences for an MG16 family enzyme.

SEQ ID NOs: 5676-5678 show peptide sequences characteristic of MG16family enzymes.

MG18

SEQ ID NOs: 1354-1511 show the full-length peptide sequences of MG18nucleases.

SEQ ID NOs: 3175-3330 show the peptide sequences of RuvC_III domains ofMG18 nucleases above.

SEQ ID NOs: 4989-5146 show the peptide of HNH domains of MG18 nucleasesabove.

SEQ ID NO: 5508 shows the nucleotide sequences of MG18 tracrRNA derivedfrom the same loci as MG18 nucleases above.

SEQ ID NOs: 5472 shows the nucleotide sequence of an sgRNA engineered tofunction with an MG18 nuclease.

SEQ ID NO: 5584 shows a nucleotide sequence for an E. colicodon-optimized coding sequences for an MG18 family enzyme.

SEQ ID NOs: 5679-5686 show peptide sequences characteristic of MG18family enzymes.

MG21

SEQ ID NOs: 1512-1655 show the full-length peptide sequences of MG21nucleases.

SEQ ID NOs: 3331-3474 show the peptide sequences of RuvC_III domains ofMG21 nucleases above.

SEQ ID NOs: 5147-5290 show the peptide of HNH domains of MG21 nucleasesabove.

SEQ ID NOs: 5509 show the nucleotide sequence of an MG21 tracrRNAderived from the same loci as MG21 nucleases above.

SEQ ID NOs: 5473 shows the nucleotide sequence of an sgRNA engineered tofunction with an MG21 nuclease.

SEQ ID NO: 5585 shows a nucleotide sequence for an E. colicodon-optimized coding sequences for an MG21 family enzyme.

SEQ ID NOs: 5687-5692 and 5674-5675 show peptide sequencescharacteristic of MG21 family enzymes.

MG22

SEQ ID NOs: 1656-1755 show the full-length peptide sequences of MG22nucleases.

SEQ ID NOs: 3475-3568 show the peptide sequences of RuvC_III domains ofMG22 nucleases above.

SEQ ID NOs: 5291-5389 show the peptide of HNH domains of MG22 nucleasesabove.

SEQ ID NO: 5510 show the nucleotide sequence of an MG22 tracrRNA derivedfrom the same loci as MG22 nucleases above.

SEQ ID NOs: 5474 shows the nucleotide sequence of an sgRNAs engineeredto function with an MG22 nuclease.

SEQ ID NO: 5586 shows a nucleotide sequence for an E. colicodon-optimized coding sequences for an MG22 family enzyme.

SEQ ID NOs: 5694-5699 show peptide sequences characteristic of MG22family enzymes.

MG23

SEQ ID NOs: 1756-1826 show the full-length peptide sequences of MG23nucleases.

SEQ ID NOs: 3569-3637 show the peptide sequences of RuvC_III domains ofMG23 nucleases above.

SEQ ID NOs: 5390-5460 show the peptide of HNH domains of MG23 nucleasesabove.

SEQ ID NO: 5511 shows the nucleotide sequences of an MG23 tracrRNAderived from the same loci as MG23 nucleases above.

SEQ ID NOs: 5475 shows the nucleotide sequence of an sgRNA engineered tofunction with an MG23 nuclease.

SEQ ID NO: 5587 shows a nucleotide sequence for an E. colicodon-optimized coding sequences for an MG23 family enzyme.

SEQ ID NOs: 5700-5717 show peptide sequences characteristic of MG23family enzymes.

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and describedherein, it will be obvious to those skilled in the art that suchembodiments are provided by way of example only. Numerous variations,changes, and substitutions may occur to those skilled in the art withoutdeparting from the invention. It should be understood that variousalternatives to the embodiments of the invention described herein may beemployed.

The practice of some methods disclosed herein employ, unless otherwiseindicated, techniques of immunology, biochemistry, chemistry, molecularbiology, microbiology, cell biology, genomics and recombinant DNA. Seefor example Sambrook and Green, Molecular Cloning: A Laboratory Manual,4th Edition (2012); the series Current Protocols in Molecular Biology(F. M. Ausubel, et al. eds.); the series Methods In Enzymology (AcademicPress, Inc.), PCR 2: A Practical Approach (M. J. MacPherson, B. D. Hamesand G. R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies,A Laboratory Manual, and Culture of Animal Cells: A Manual of BasicTechnique and Specialized Applications, 6th Edition (R. I. Freshney, ed.(2010)) (which is entirely incorporated by reference herein).

As used herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. Furthermore, to the extent that the terms “including”,“includes”, “having”, “has”, “with”, or variants thereof are used ineither the detailed description and/or the claims, such terms areintended to be inclusive in a manner similar to the term “comprising”.

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” can mean within one or more than one standarddeviation, per the practice in the art. Alternatively, “about” can meana range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of agiven value.

As used herein, a “cell” generally refers to a biological cell. A cellmay be the basic structural, functional and/or biological unit of aliving organism. A cell may originate from any organism having one ormore cells. Some non-limiting examples include: a prokaryotic cell,eukaryotic cell, a bacterial cell, an archaeal cell, a cell of asingle-cell eukaryotic organism, a protozoa cell, a cell from a plant(e.g., cells from plant crops, fruits, vegetables, grains, soy bean,corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin,hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers,gymnosperms, ferns, clubmosses, homworts, liverworts, mosses), an algalcell, (e.g., Botryococcus braunii, Chlamydomonas reinhardtii,Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens C.Agardh, and the like), seaweeds (e.g., kelp), a fungal cell (e.g., ayeast cell, a cell from a mushroom), an animal cell, a cell from aninvertebrate animal (e.g., fruit fly, cnidarian, echinoderm, nematode,etc.), a cell from a vertebrate animal (e.g., fish, amphibian, reptile,bird, mammal), a cell from a mammal (e.g., a pig, a cow, a goat, asheep, a rodent, a rat, a mouse, a non-human primate, a human, etc.),and etcetera. Sometimes a cell is not originating from a naturalorganism (e.g., a cell can be a synthetically made, sometimes termed anartificial cell).

The term “nucleotide,” as used herein, generally refers to abase-sugar-phosphate combination. A nucleotide may comprise a syntheticnucleotide. A nucleotide may comprise a synthetic nucleotide analog.Nucleotides may be monomeric units of a nucleic acid sequence (e.g.,deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The termnucleotide may include ribonucleoside triphosphates adenosinetriphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate(CTP), guanosine triphosphate (GTP) and deoxyribonucleosidetriphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivativesthereof. Such derivatives may include, for example, [αS]dATP,7-deaza-dGTP and 7-deaza-dATP, and nucleotide derivatives that confernuclease resistance on the nucleic acid molecule containing them. Theterm nucleotide as used herein may refer to dideoxyribonucleosidetriphosphates (ddNTPs) and their derivatives. Illustrative examples ofdideoxyribonucleoside triphosphates may include, but are not limited to,ddATP, ddCTP, ddGTP, ddITP, and ddTTP. A nucleotide may be unlabeled ordetectably labeled, such as using moieties comprising opticallydetectable moieties (e.g., fluorophores). Labeling may also be carriedout with quantum dots. Detectable labels may include, for example,radioactive isotopes, fluorescent labels, chemiluminescent labels,bioluminescent labels and enzyme labels. Fluorescent labels ofnucleotides may include but are not limited fluorescein,5-carboxyfluorescein (FAM),2′7′-dimethoxy-4′5-dichloro-6-carboxyfluorescein (JOE), rhodamine,6-carboxyrhodamine (R6G), N,N,N′,N′-tetramethyl-6-carboxyrhodamine(TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4′dimethylaminophenylazo)benzoic acid (DABCYL), Cascade Blue, Oregon Green, Texas Red, Cyanineand 5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS). Specificexamples of fluorescently labeled nucleotides can include [R6G]dUTP,[TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP,[FAM]ddCTP, [R110]ddCTP, [TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP,[dR110]ddCTP, [dTAMRA]ddGTP, and [dROX]ddTTP available from PerkinElmer, Foster City, Calif.; FluoroLink DeoxyNucleotides, FluoroLinkCy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLinkCy3-dUTP, and FluoroLink Cy5-dUTP available from Amersham, ArlingtonHeights, Ill.; Fluorescein-15-dATP, Fluorescein-12-dUTP,Tetramethyl-rodamine-6-dUTP, IR770-9-dATP, Fluorescein-12-ddUTP,Fluorescein-12-UTP, and Fluorescein-15-2′-dATP available from BoehringerMannheim, Indianapolis, Ind.; and Chromosome Labeled Nucleotides,BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP,BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, CascadeBlue-7-UTP, Cascade Blue-7-dUTP, fluorescein-12-UTP,fluorescein-12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP,Rhodamine Green-5-dUTP, tetramethylrhodamine-6-UTP,tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, andTexas Red-12-dUTP available from Molecular Probes, Eugene, Oreg.Nucleotides can also be labeled or marked by chemical modification. Achemically-modified single nucleotide can be biotin-dNTP. Somenon-limiting examples of biotinylated dNTPs can include, biotin-dATP(e.g., bio-N6-ddATP, biotin-14-dATP), biotin-dCTP (e.g., biotin-11-dCTP,biotin-14-dCTP), and biotin-dUTP (e.g., biotin-11-dUTP, biotin-16-dUTP,biotin-20-dUTP).

The terms “polynucleotide,” “oligonucleotide,” and “nucleic acid” areused interchangeably to generally refer to a polymeric form ofnucleotides of any length, either deoxyribonucleotides orribonucleotides, or analogs thereof, either in single-, double-, ormulti-stranded form. A polynucleotide may be exogenous or endogenous toa cell. A polynucleotide may exist in a cell-free environment. Apolynucleotide may be a gene or fragment thereof. A polynucleotide maybe DNA. A polynucleotide may be RNA. A polynucleotide may have anythree-dimensional structure and may perform any function. Apolynucleotide may comprise one or more analogs (e.g., altered backbone,sugar, or nucleobase). If present, modifications to the nucleotidestructure may be imparted before or after assembly of the polymer. Somenon-limiting examples of analogs include: 5-bromouracil, peptide nucleicacid, xeno nucleic acid, morpholinos, locked nucleic acids, glycolnucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin,7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to thesugar), thiol containing nucleotides, biotin linked nucleotides,fluorescent base analogs, CpG islands, methyl-7-guanosine, methylatednucleotides, inosine, thiouridine, pseudourdine, dihydrouridine,queuosine, and wyosine. Non-limiting examples of polynucleotides includecoding or non-coding regions of a gene or gene fragment, loci (locus)defined from linkage analysis, exons, introns, messenger RNA (mRNA),transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA(siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA,recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,cell-free polynucleotides including cell-free DNA (cfDNA) and cell-freeRNA (cfRNA), nucleic acid probes, and primers. The sequence ofnucleotides may be interrupted by non-nucleotide components.

The terms “transfection” or “transfected” generally refer tointroduction of a nucleic acid into a cell by non-viral or viral-basedmethods. The nucleic acid molecules may be gene sequences encodingcomplete proteins or functional portions thereof. See, e.g., Sambrook etal., 1989, Molecular Cloning: A Laboratory Manual, 18.1-18.88.

The terms “peptide,” “polypeptide,” and “protein” are usedinterchangeably herein to generally refer to a polymer of at least twoamino acid residues joined by peptide bond(s). This term does notconnote a specific length of polymer, nor is it intended to imply ordistinguish whether the peptide is produced using recombinanttechniques, chemical or enzymatic synthesis, or is naturally occurring.The terms apply to naturally occurring amino acid polymers as well asamino acid polymers comprising at least one modified amino acid. In somecases, the polymer may be interrupted by non-amino acids. The termsinclude amino acid chains of any length, including full length proteins,and proteins with or without secondary and/or tertiary structure (e.g.,domains). The terms also encompass an amino acid polymer that has beenmodified, for example, by disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, oxidation, and any othermanipulation such as conjugation with a labeling component. The terms“amino acid” and “amino acids,” as used herein, generally refer tonatural and non-natural amino acids, including, but not limited to,modified amino acids and amino acid analogues. Modified amino acids mayinclude natural amino acids and non-natural amino acids, which have beenchemically modified to include a group or a chemical moiety notnaturally present on the amino acid. Amino acid analogues may refer toamino acid derivatives. The term “amino acid” includes both D-aminoacids and L-amino acids.

As used herein, the “non-native” can generally refer to a nucleic acidor polypeptide sequence that is not found in a native nucleic acid orprotein. Non-native may refer to affinity tags. Non-native may refer tofusions. Non-native may refer to a naturally occurring nucleic acid orpolypeptide sequence that comprises mutations, insertions and/ordeletions. A non-native sequence may exhibit and/or encode for anactivity (e.g., enzymatic activity, methyltransferase activity,acetyltransferase activity, kinase activity, ubiquitinating activity,etc.) that may also be exhibited by the nucleic acid and/or polypeptidesequence to which the non-native sequence is fused. A non-native nucleicacid or polypeptide sequence may be linked to a naturally-occurringnucleic acid or polypeptide sequence (or a variant thereof) by geneticengineering to generate a chimeric nucleic acid and/or polypeptidesequence encoding a chimeric nucleic acid and/or polypeptide.

The term “promoter”, as used herein, generally refers to the regulatoryDNA region which controls transcription or expression of a gene andwhich may be located adjacent to or overlapping a nucleotide or regionof nucleotides at which RNA transcription is initiated. A promoter maycontain specific DNA sequences which bind protein factors, oftenreferred to as transcription factors, which facilitate binding of RNApolymerase to the DNA leading to gene transcription. A ‘basal promoter’,also referred to as a ‘core promoter’, may generally refer to a promoterthat contains all the basic necessary elements to promotetranscriptional expression of an operably linked polynucleotide.Eukaryotic basal promoters typically, though not necessarily, contain aTATA-box and/or a CAAT box.

The term “expression”, as used herein, generally refers to the processby which a nucleic acid sequence or a polynucleotide is transcribed froma DNA template (such as into mRNA or other RNA transcript) and/or theprocess by which a transcribed mRNA is subsequently translated intopeptides, polypeptides, or proteins. Transcripts and encodedpolypeptides may be collectively referred to as “gene product.” If thepolynucleotide is derived from genomic DNA, expression may includesplicing of the mRNA in a eukaryotic cell.

As used herein, “operably linked”, “operable linkage”, “operativelylinked”, or grammatical equivalents thereof generally refer tojuxtaposition of genetic elements, e.g., a promoter, an enhancer, apolyadenylation sequence, etc., wherein the elements are in arelationship permitting them to operate in the expected manner. Forinstance, a regulatory element, which may comprise promoter and/orenhancer sequences, is operatively linked to a coding region if theregulatory element helps initiate transcription of the coding sequence.There may be intervening residues between the regulatory element andcoding region so long as this functional relationship is maintained.

A “vector” as used herein, generally refers to a macromolecule orassociation of macromolecules that comprises or associates with apolynucleotide and which may be used to mediate delivery of thepolynucleotide to a cell. Examples of vectors include plasmids, viralvectors, liposomes, and other gene delivery vehicles. The vectorgenerally comprises genetic elements, e.g., regulatory elements,operatively linked to a gene to facilitate expression of the gene in atarget.

As used herein, “an expression cassette” and “a nucleic acid cassette”are used interchangeably generally to refer to a combination of nucleicacid sequences or elements that are expressed together or are operablylinked for expression. In some cases, an expression cassette refers tothe combination of regulatory elements and a gene or genes to which theyare operably linked for expression.

A “functional fragment” of a DNA or protein sequence generally refers toa fragment that retains a biological activity (either functional orstructural) that is substantially similar to a biological activity ofthe full-length DNA or protein sequence. A biological activity of a DNAsequence may be its ability to influence expression in a manner known tobe attributed to the full-length sequence.

As used herein, an “engineered” object generally indicates that theobject has been modified by human intervention. According tonon-limiting examples: a nucleic acid may be modified by changing itssequence to a sequence that does not occur in nature; a nucleic acid maybe modified by ligating it to a nucleic acid that it does not associatewith in nature such that the ligated product possesses a function notpresent in the original nucleic acid; an engineered nucleic acid maysynthesized in vitro with a sequence that does not exist in nature; aprotein may be modified by changing its amino acid sequence to asequence that does not exist in nature; an engineered protein mayacquire a new function or property. An “engineered” system comprises atleast one engineered component.

As used herein, “synthetic” and “artificial” are used interchangeably torefer to a protein or a domain thereof that has low sequence identity(e.g., less than 50% sequence identity, less than 25% sequence identity,less than 10% sequence identity, less than 5% sequence identity, lessthan 1% sequence identity) to a naturally occurring human protein. Forexample, VPR and VP64 domains are synthetic transactivation domains.

The term “tracrRNA” or “tracr sequence”, as used herein, can generallyrefer to a nucleic acid with at least about 5%, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, or 100% sequence identity and/or sequencesimilarity to a wild type exemplary tracrRNA sequence (e.g., a tracrRNAfrom S. pyogenes S. aureus, etc or SEQ ID NOs: 5476-5511). tracrRNA canrefer to a nucleic acid with at most about 5%, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, or 100% sequence identity and/or sequence similarityto a wild type exemplary tracrRNA sequence (e.g., a tracrRNA from S.pyogenes S. aureus, etc). tracrRNA may refer to a modified form of atracrRNA that can comprise a nucleotide change such as a deletion,insertion, or substitution, variant, mutation, or chimera. A tracrRNAmay refer to a nucleic acid that can be at least about 60% identical toa wild type exemplary tracrRNA (e.g., a tracrRNA from S. pyogenes S.aureus, etc) sequence over a stretch of at least 6 contiguousnucleotides. For example, a tracrRNA sequence can be at least about 60%identical, at least about 65% identical, at least about 70% identical,at least about 75% identical, at least about 80% identical, at leastabout 85% identical, at least about 90% identical, at least about 95%identical, at least about 98% identical, at least about 99% identical,or 100% identical to a wild type exemplary tracrRNA (e.g., a tracrRNAfrom S. pyogenes S. aureus, etc) sequence over a stretch of at least 6contiguous nucleotides. Type II tracrRNA sequences can be predicted on agenome sequence by identifying regions with complementarity to part ofthe repeat sequence in an adjacent CRISPR array.

As used herein, a “guide nucleic acid” can generally refer to a nucleicacid that may hybridize to another nucleic acid. A guide nucleic acidmay be RNA. A guide nucleic acid may be DNA. The guide nucleic acid maybe programmed to bind to a sequence of nucleic acid site-specifically.The nucleic acid to be targeted, or the target nucleic acid, maycomprise nucleotides. The guide nucleic acid may comprise nucleotides. Aportion of the target nucleic acid may be complementary to a portion ofthe guide nucleic acid. The strand of a double-stranded targetpolynucleotide that is complementary to and hybridizes with the guidenucleic acid may be called the complementary strand. The strand of thedouble-stranded target polynucleotide that is complementary to thecomplementary strand, and therefore may not be complementary to theguide nucleic acid may be called noncomplementary strand. A guidenucleic acid may comprise a polynucleotide chain and can be called a“single guide nucleic acid.” A guide nucleic acid may comprise twopolynucleotide chains and may be called a “double guide nucleic acid.”If not otherwise specified, the term “guide nucleic acid” may beinclusive, referring to both single guide nucleic acids and double guidenucleic acids. A guide nucleic acid may comprise a segment that can bereferred to as a “nucleic acid-targeting segment” or a “nucleicacid-targeting sequence.” A nucleic acid-targeting segment may comprisea sub-segment that may be referred to as a “protein binding segment” or“protein binding sequence” or “Cas protein binding segment”.

The term “sequence identity” or “percent identity” in the context of twoor more nucleic acids or polypeptide sequences, generally refers to two(e.g., in a pairwise alignment) or more (e.g., in a multiple sequencealignment) sequences that are the same or have a specified percentage ofamino acid residues or nucleotides that are the same, when compared andaligned for maximum correspondence over a local or global comparisonwindow, as measured using a sequence comparison algorithm. Suitablesequence comparison algorithms for polypeptide sequences include, e.g.,BLASTP using parameters of a wordlength (W) of 3, an expectation (E) of10, and the BLOSUM62 scoring matrix setting gap costs at existence of11, extension of 1, and using a conditional compositional score matrixadjustment for polypeptide sequences longer than 30 residues; BLASTPusing parameters of a wordlength (W) of 2, an expectation (E) of1000000, and the PAM30 scoring matrix setting gap costs at 9 to opengaps and 1 to extend gaps for sequences of less than 30 residues (theseare the default parameters for BLASTP in the BLAST suite available atwww.blast.ncbi.nlm.nih.gov); CLUSTALW with parameters of; theSmith-Waterman homology search algorithm with parameters of a match of2, a mismatch of −1, and a gap of −1; MUSCLE with default parameters;MAFFT with parameters retree of 2 and maxiterations of 1000; Novafoldwith default parameters; HMMER hmmalign with default parameters.

Included in the current disclosure are variants of any of the enzymedescribed herein with one or more conservative amino acid substitutions.Such conservative substitutions can be made in the amino acid sequenceof a polypeptide without disrupting the three-dimensional structure orfunction of the polypeptide. Conservative substitutions can beaccomplished by substituting amino acids with similar hydrophobicity,polarity, and R chain length for one another. Additionally oralternatively, by comparing aligned sequences of homologous proteinsfrom different species, conservative substitutions can be identified bylocating amino acid residues that have been mutated between species(e.g. non-conserved residues) without altering the basic functions ofthe encoded proteins. Such conservatively substituted variants mayinclude variants with at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 55%, at least about 60%, at leastabout 65%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 91%, at leastabout 92%, at least about 93%, at least about 94%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, at leastabout 99% identity to any one of the endonuclease protein sequencesdescribed herein (e.g. MG1, MG2, MG3, MG4, MG6, MG7, MG14, MG15, MG16,MG18, MG21, MG22, or MG23 family endonucleases described herein). Insome embodiments, such conservatively substituted variants arefunctional variants. Such functional variants can encompass sequenceswith substitutions such that the activity of critical active siteresidues of the endonuclease are not disrupted.

Conservative substitution tables providing functionally similar aminoacids are available from a variety of references (see, for e.g.,Creighton, Proteins: Structures and Molecular Properties (W H Freeman &Co.; 2nd edition (December 1993)). The following eight groups eachcontain amino acids that are conservative substitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5)Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S),Threonine (T); and 8) Cysteine (C), Methionine (M).

As used herein, the term “RuvC_III domain” generally refers to a thirddiscontinuous segment of a RuvC endonuclease domain (the RuvC nucleasedomain being comprised of three discontiguous segments, RuvC_I, RuvC_II,and RuvC_III). A RuvC domain or segments thereof can generally beidentified by alignment to known domain sequences, structural alignmentto proteins with annotated domains, or by comparison to Hidden MarkovModels (HMMs) built based on known domain sequences (e.g., Pfam HMMPF18541 for RuvC_III).

As used herein, the term “HNH domain” generally refers to anendonuclease domain having characteristic histidine and asparagineresidues. An HNH domain can generally be identified by alignment toknown domain sequences, structural alignment to proteins with annotateddomains, or by comparison to Hidden Markov Models (HMMs) built based onknown domain sequences (e.g., Pfam HMM PF01844 for domain HNH).

Overview

The discovery of new Cas enzymes with unique functionality and structuremay offer the potential to further disrupt deoxyribonucleic acid (DNA)editing technologies, improving speed, specificity, functionality, andease of use. Relative to the predicted prevalence of Clustered RegularlyInterspaced Short Palindromic Repeats (CRISPR) systems in microbes andthe sheer diversity of microbial species, relatively few functionallycharacterized CRISPR/Cas enzymes exist in the literature. This is partlybecause a huge number of microbial species may not be readily cultivatedin laboratory conditions. Metagenomic sequencing from naturalenvironmental niches that represent large numbers of microbial speciesmay offer the potential to drastically increase the number of newCRISPR/Cas systems known and speed the discovery of new oligonucleotideediting functionalities. A recent example of the fruitfulness of such anapproach is demonstrated by the 2016 discovery of CasX/CasY CRISPRsystems from metagenomic analysis of natural microbial communities.

CRISPR/Cas systems are RNA-directed nuclease complexes that have beendescribed to function as an adaptive immune system in microbes. In theirnatural context, CRISPR/Cas systems occur in CRISPR (clustered regularlyinterspaced short palindromic repeats) operons or loci, which generallycomprise two parts: (i) an array of short repetitive sequences (30-40bp) separated by equally short spacer sequences, which encode theRNA-based targeting element; and (ii) ORFs encoding the Cas encoding thenuclease polypeptide directed by the RNA-based targeting elementalongside accessory proteins/enzymes. Efficient nuclease targeting of aparticular target nucleic acid sequence generally requires both (i)complementary hybridization between the first 6-8 nucleic acids of thetarget (the target seed) and the crRNA guide; and (ii) the presence of aprotospacer-adjacent motif (PAM) sequence within a defined vicinity ofthe target seed (the PAM usually being a sequence not commonlyrepresented within the host genome). Depending on the exact function andorganization of the system, CRISPR-Cas systems are commonly organizedinto 2 classes, 5 types and 16 subtypes based on shared functionalcharacteristics and evolutionary similarity.

Class I CRISPR-Cas systems have large, multisubunit effector complexes,and comprise Types I, III, and IV.

Type I CRISPR-Cas systems are considered of moderate complexity in termsof components. In Type I CRISPR-Cas systems, the array of RNA-targetingelements is transcribed as a long precursor crRNA (pre-crRNA) that isprocessed at repeat elements to liberate short, mature crRNAs thatdirect the nuclease complex to nucleic acid targets when they arefollowed by a suitable short consensus sequence called aprotospacer-adjacent motif (PAM). This processing occurs via anendoribonuclease subunit (Cas6) of a large endonuclease complex calledCascade, which also comprises a nuclease (Cas3) protein component of thecrRNA-directed nuclease complex. Cas I nucleases function primarily asDNA nucleases.

Type III CRISPR systems may be characterized by the presence of acentral nuclease, known as Cas10, alongside a repeat-associatedmysterious protein (RAMP) that comprises Csm or Cmr protein subunits.Like in Type I systems, the mature crRNA is processed from a pre-crRNAusing a Cas6-like enzyme. Unlike type I and II systems, type III systemsappear to target and cleave DNA-RNA duplexes (such as DNA strands beingused as templates for an RNA polymerase).

Type IV CRISPR-Cas systems possess an effector complex that consists ofa highly reduced large subunit nuclease (csf1), two genes for RAMPproteins of the Cas5 (csf3) and Cas7 (csf2) groups, and, in some cases,a gene for a predicted small subunit; such systems are commonly found onendogenous plasmids.

Class II CRISPR-Cas systems generally have single-polypeptidemultidomain nuclease effectors, and comprise Types II, V and VI.

Type II CRISPR-Cas systems are considered the simplest in terms ofcomponents. In Type II CRISPR-Cas systems, the processing of the CRISPRarray into mature crRNAs does not require the presence of a specialendonuclease subunit, but rather a small trans-encoded crRNA (tracrRNA)with a region complementary to the array repeat sequence; the tracrRNAinteracts with both its corresponding effector nuclease (e.g. Cas9) andthe repeat sequence to form a precursor dsRNA structure, which iscleaved by endogenous RNAse III to generate a mature effector enzymeloaded with both tracrRNA and crRNA. Cas II nucleases are known as DNAnucleases. Type 2 effectors generally exhibit a structure consisting ofa RuvC-like endonuclease domain that adopts the RNase H fold with anunrelated HNH nuclease domain inserted within the folds of the RuvC-likenuclease domain. The RuvC-like domain is responsible for the cleavage ofthe target (e.g., crRNA complementary) DNA strand, while the HNH domainis responsible for cleavage of the displaced DNA strand.

Type V CRISPR-Cas systems are characterized by a nuclease effector (e.g.Cas12) structure similar to that of Type II effectors, comprising aRuvC-like domain. Similar to Type II, most (but not all) Type V CRISPRsystems use a tracrRNA to process pre-crRNAs into mature crRNAs;however, unlike Type II systems which requires RNAse III to cleave thepre-crRNA into multiple crRNAs, type V systems are capable of using theeffector nuclease itself to cleave pre-crRNAs. Like Type-II CRISPR-Cassystems, Type V CRISPR-Cas systems are again known as DNA nucleases.Unlike Type II CRISPR-Cas systems, some Type V enzymes (e.g., Cas12a)appear to have a robust single-stranded nonspecific deoxyribonucleaseactivity that is activated by the first crRNA directed cleavage of adouble-stranded target sequence.

Type VI CRIPSR-Cas systems have RNA-guided RNA endonucleases. Instead ofRuvC-like domains, the single polypeptide effector of Type VI systems(e.g. Cas13) comprises two HEPN ribonuclease domains. Differing fromboth Type II and V systems, Type VI systems also appear to not need atracrRNA for processing of pre-crRNA into crRNA. Similar to type Vsystems, however, some Type VI systems (e.g., C2C2) appear to possessrobust single-stranded nonspecific nuclease (ribonuclease) activityactivated by the first crRNA directed cleavage of a target RNA.

Because of their simpler architecture, Class II CRISPR-Cas have beenmost widely adopted for engineering and development as designernuclease/genome editing applications.

One of the early adaptations of such a system for in vitro use can befound in Jinek et al. (Science. 2012 Aug. 17; 337(6096):816-21, which isentirely incorporated herein by reference). The Jinek study firstdescribed a system that involved (i) recombinantly-expressed, purifiedfull-length Cas9 (e.g., a Class II, Type II Cas enzyme) isolated from S.pyogenes SF370, (ii) purified mature ˜42 nt crRNA bearing a ˜20 nt 5′sequence complementary to the target DNA sequence desired to be cleavedfollowed by a 3′ tracr-binding sequence (the whole crRNA being in vitrotranscribed from a synthetic DNA template carrying a T7 promotersequence); (iii) purified tracrRNA in vitro transcribed from a syntheticDNA template carrying a T7 promoter sequence, and (iv) Mg²⁺. Jinek laterdescribed an improved, engineered system wherein the crRNA of (ii) isjoined to the 5′ end of (iii) by a linker (e.g., GAAA) to form a singlefused synthetic guide RNA (sgRNA) capable of directing Cas9 to a targetby itself (compare top and bottom panel of FIG. 2).

Mali et al. (Science. 2013 Feb. 15; 339(6121): 823-826.), which isentirely incorporated herein by reference, later adapted this system foruse in mammalian cells by providing DNA vectors encoding (i) an ORFencoding codon-optimized Cas9 (e.g., a Class II, Type II Cas enzyme)under a suitable mammalian promoter with a C-terminal nuclearlocalization sequence (e.g., SV40 NLS) and a suitable polyadenylationsignal (e.g., TK pA signal); and (ii) an ORF encoding an sgRNA (having a5′ sequence beginning with G followed by 20 nt of a complementarytargeting nucleic acid sequence joined to a 3′ tracr-binding sequence, alinker, and the tracrRNA sequence) under a suitable Polymerase IIIpromoter (e.g., the U6 promoter).

MG1 Enzymes

In one aspect, the present disclosure provides for an engineerednuclease system comprising (a) an endonuclease. In some cases, theendonuclease is a Cas endonuclease. In some cases, the endonuclease is aType II, Class II Cas endonuclease. The endonuclease may comprise aRuvC_III domain, wherein said RuvC_III domain has at least about 70%sequence identity to any one of SEQ ID NOs: 1827-2140. In some cases,the endonuclease may comprise a RuvC_III domain, wherein the RuvC_IIIdomain has at least about 20%, at least about 25%, at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 91%, at least about 92%,at least about 93%, at least about 94%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, at least about 99%identity to any one of SEQ ID NOs: 1827-2140. In some cases, theendonuclease may comprise a RuvC_III domain, wherein the substantiallyidentical to any one of SEQ ID NOs: 1827-2140. The endonuclease maycomprise a RuvC_III domain having at least about 70% sequence identityto any one of SEQ ID NOs: 1827-1831. In some cases, the endonuclease maycomprise a RuvC_III domain having at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about91%, at least about 92%, at least about 93%, at least about 94%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, at least about 99% identity to any one of SEQ ID NOs: 1827-1831. Insome cases, the endonuclease may comprise a RuvC_III domainsubstantially identical to any one of SEQ ID NOs: 1827-1831. In somecases, the endonuclease may comprise a RuvC_III domain having at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 91%, at least about 92%, at leastabout 93%, at least about 94%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, at least about 99% identity toSEQ ID NO: 1827. In some cases, the endonuclease may comprise a RuvC_IIIdomain having at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 91%, atleast about 92%, at least about 93%, at least about 94%, at least about95%, at least about 96%, at least about 97%, at least about 98%, atleast about 99% identity to SEQ ID NO: 1828. In some cases, theendonuclease may comprise a RuvC_III domain having at least about 70%,at least about 75%, at least about 80%, at least about 85%, at leastabout 90%, at least about 91%, at least about 92%, at least about 93%,at least about 94%, at least about 95%, at least about 96%, at leastabout 97%, at least about 98%, at least about 99% identity to SEQ ID NO:1829. In some cases, the endonuclease may comprise a RuvC_III domainhaving at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, at least about99% identity to SEQ ID NO: 1830. In some cases, the endonuclease maycomprise a RuvC_III domain having at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 91%, at least about 92%, at least about 93%, at least about94%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, at least about 99% identity to SEQ ID NO: 1831.

The endonuclease may comprise an HNH domain having at least about 70%identity to any one of SEQ ID NOs: 3638-3955. In some cases, theendonuclease may comprise an HNH domain having at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% identical to any one ofSEQ ID NOs: 3638-3955. The endonuclease may comprise an HNH domainsubstantially identical to any one of SEQ ID NOs: 3638-3955. Theendonuclease may comprise an HNH domain having at least about 70%identity to any one of SEQ ID NOs: 3638-3955. In some cases, theendonuclease may comprise an HNH domain having at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% identical to any one ofSEQ ID NOs: 3638-3955. The endonuclease may comprise an HNH domainsubstantially identical to any one of SEQ ID NOs: 3638-3955. Theendonuclease may comprise an HNH domain having at least about 70%identity to any one of SEQ ID NOs: 3638-3641. In some cases, theendonuclease may comprise an HNH domain having at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% identical to any one ofSEQ ID NOs: 3638-3641. The endonuclease may comprise an HNH domainsubstantially identical to any one of SEQ ID NOs: 3638-3641. Theendonuclease may comprise an HNH domain having at least about 70%identity to any one of SEQ ID NOs: 3638. In some cases, the endonucleasemay comprise an HNH domain having at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 91%, at least about 92%, at least about 93%, at least about94%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, or at least about 99% identical to any one of SEQ IDNOs: 3638. The endonuclease may comprise an HNH domain substantiallyidentical to any one of SEQ ID NOs: 3638. The endonuclease may comprisean HNH domain having at least about 70% identity to any one of SEQ IDNOs: 3639. In some cases, the endonuclease may comprise an HNH domainhaving at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, or at leastabout 99% identical to any one of SEQ ID NOs: 3639. The endonuclease maycomprise an HNH domain substantially identical to any one of SEQ ID NOs:3639. The endonuclease may comprise an HNH domain having at least about70% identity to any one of SEQ ID NOs: 3640. In some cases, theendonuclease may comprise an HNH domain having at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% identical to any one ofSEQ ID NOs: 3640. The endonuclease may comprise an HNH domainsubstantially identical to any one of SEQ ID NOs: 3640. The endonucleasemay comprise an HNH domain having at least about 70% identity to any oneof SEQ ID NOs: 3641. In some cases, the endonuclease may comprise an HNHdomain having at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 91%, atleast about 92%, at least about 93%, at least about 94%, at least about95%, at least about 96%, at least about 97%, at least about 98%, or atleast about 99% identical to any one of SEQ ID NOs: 3641. Theendonuclease may comprise an HNH domain substantially identical to anyone of SEQ ID NOs: 3641.

In some cases, the endonuclease may comprise a variant having at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 55%, at least about 60%, at leastabout 65%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, or at least about 99%identity to any one of SEQ ID NOs: 1-6 or 9-319. In some cases, theendonuclease may be substantially identical to any one of SEQ ID NOs:1-6 or 9-319. In some cases, the endonuclease may comprise a varianthaving at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 96%, at least about 97%, at least about 98%, or atleast about 99% identity to any one of SEQ ID NOs:1-4. In some cases,the endonuclease may be substantially identical to any one of SEQ IDNOs: 1-4. In some cases, the endonuclease may comprise a peptide motifsubstantially identical to any one of SEQ ID NOs: 5615, 5616, or 5617.

In some cases, the endonuclease may comprise a variant having one ormore nuclear localization sequences (NLSs). The NLS may be proximal tothe N- or C-terminus of said endonuclease. The NLS may be appendedN-terminal or C-terminal to any one of SEQ ID NOs: 1-6 or 9-319, or to avariant having at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 55%, atleast about 60%, at least about 65%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99% identity to any one of SEQ ID NOs: 1-319. TheNLS may be an SV40 large T antigen NLS. The NLS may be a c-myc NLS. TheNLS can comprise a sequence with at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 99% identity toany one of SEQ ID NOs: 5593-5608. The NLS can comprise a sequencesubstantially identical to any one of SEQ ID NOs: 5593-5608. The NLS cancomprise any of the sequences in Table 1 below, or a combinationthereof:

TABLE 1Example NLS Sequences that can be used with Cas Effectors According tothe Disclosure Source NLS amino acid sequence SEQ ID NO: SV40 PKKKRKV5597 nucleoplasmin bipartite NLS LKRPAATKKAGQAKKKK 5598 c-myc NLSPAAKRVKLD 5599 c-myc NLS RQRRNELKRSP 5600 hRNPA1 M9NLSNQSSNFGPMKGGNFGGRSSGPYGGGGQYFA 5601 KPRNQGGY Importin-alpha IBB domainRMRIZFKNKGKDTAELRRRRVEVSVELRKAK 5602 KDEQILKRRNV Myoma T proteinVSRKRPRP 5603 Myoma T protein PPKKARED 5604 p53 PQPKKKPL 5605mouse c-ab1 IV SALIKKKKKMAP 5606 influenza virus NS1 DRLRR 5607influenza virus NS1 PKQKKRK 5608 Hepatitis virus delta antigenRKLKKKIKKL 5609 mouse Mx1 protein REKKKFLKRR 5610 human poly(ADP-ribose)KRKGDEVDGVDEVAKKKSKK 5611 polymerase steroid hormone receptorRKCLQAGMNLEARKTKK 5612 (human) glucocorticoid

In some cases, the endonuclease may be recombinant (e.g., cloned,expressed, and purified by a suitable method such as expression in E.coli followed by epitope-tag purification). In some cases, theendonuclease may be derived from a bacterium with a 16S rRNA gene havingat least about 90% identity to any one of SEQ ID NOs: 5592-5595. Theendonuclease may be derived from a species having a 16S rRNA gene atleast about 80%, at least about 82%, at least about 83%, at least about84%, at least about 85%, at least about 86%, at least about 87%, atleast about 88%, at least about 89%, at least about 90%, at least about91%, at least about 92%, at least about 93%, at least about 94%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99% identity to any one of SEQ ID NOs: 5592-5595.The endonuclease may be derived from a species having a 16S rRNA genesubstantially identical to any one of SEQ ID NOs: 5592-5595. Theendonuclease may be derived from a bacterium belonging to the PhylumVerrucomicrobia or the Phylum Candidatus Peregrinibacteria.

In some cases, sequence identity may be determined by the BLASTP,CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology searchalgorithm. The sequence identity may be determined by the BLASTPalgorithm using parameters of a wordlength (W) of 3, an expectation (E)of 10, and using a BLOSUM62 scoring matrix setting gap costs atexistence of 11, extension of 1, and using a conditional compositionalscore matrix adjustment.

In some cases, the system above may comprise (b) at least one engineeredsynthetic guide ribonucleic acid (sgRNA) capable of forming a complexwith the endonuclease bearing a 5′ targeting region complementary to adesired cleavage sequence. In some cases, the 5′ targeting region maycomprises a PAM sequence compatible with the endonuclease. In somecases, the 5′ most nucleotide of the targeting region may be G. In somecases, the 5′ targeting region may be 15-23 nucleotides in length. Theguide sequence and the tracr sequence may be supplied as separateribonucleic acids (RNAs) or a single ribonucleic acid (RNA). The guideRNA may comprise a crRNA tracrRNA binding sequence 3′ to the targetingregion. The guide RNA may comprise a tracrRNA sequence preceded by a4-nucleotide linker 3′ to the crRNA tracrRNA binding region. The sgRNAmay comprise, from 5′ to 3′: a non-natural guide nucleic acid sequencecapable of hybridizing to a target sequence in a cell; and a tracrsequence. In some cases, the non-natural guide nucleic acid sequence andthe tracr sequence are covalently linked.

In some cases, the tracr sequence may have a particular sequence. Thetracr sequence may have at least about 80% to at least about 60-100(e.g., at least about 60, at least about 65, at least about 70, at leastabout 75, at least about 80, at least about 85, or at least about 90)consecutive nucleotides of a natural tracrRNA sequence. The tracrsequence may have at least about 80% sequence identity to at least about60-100 (e.g., at least about 60, at least about 65, at least about 70,at least about 75, at least about 80, at least about 85, or at leastabout 90) consecutive nucleotides of any one of SEQ ID NOs: 5476-5489.In some cases, the tracrRNA may have at least about 80%, at least about85%, at least about 90%, at least about 91%, at least about 92%, atleast about 93%, at least about 94%, at least about 95%, at least about96%, at least about 97%, at least about 98%, or at least about 99%identity to at least about 60-90 (e.g., at least about 60, at leastabout 65, at least about 70, at least about 75, at least about 80, atleast about 85, or at least about 90) consecutive nucleotides of any oneof SEQ ID NOs: 5476-5489. In some cases, the tracrRNA may besubstantially identical to at least about 60-100 (e.g., at least about60, at least about 65, at least about 70, at least about 75, at leastabout 80, at least about 85, or at least about 90) consecutivenucleotides of any one of SEQ ID NOs: 5476-5489. The tracrRNA maycomprise any of SEQ ID NOs: 5476-5489.

In some cases, the at least one engineered synthetic guide ribonucleicacid (sgRNA) capable of forming a complex with the endonuclease maycomprise a sequence having at least about 80% identity to any one of SEQID NOs: 5461-5464. The sgRNA may comprise a sequence having at leastabout 80%, at least about 85%, at least about 90%, at least about 91%,at least about 92%, at least about 93%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99% identity to any one of SEQ ID NOs: 5461-5464. ThesgRNA may comprise a sequence substantially identical to any one of SEQID NOs: 5461-5464.

In some cases, the system above may comprise two different sgRNAstargeting a first region and a second region for cleavage in a targetDNA locus, wherein the second region is 3′ to the first region. In somecases, the system above may comprise a single- or double-stranded DNArepair template comprising from 5′ to 3′: a first homology armcomprising a sequence of at least about 20 (e.g., at least about 40, 80,120, 150, 200, 300, 500, or 1 kb) nucleotides 5′ to the first region, asynthetic DNA sequence of at least about 10 nucleotides, and a secondhomology arm comprising a sequence of at least about 20 (e.g., at leastabout 40, 80, 120, 150, 200, 300, 500, or 1 kb) nucleotides 3′ to thesecond region.

In another aspect, the present disclosure provides a method formodifying a target nucleic acid locus. The method may comprisedelivering to the target nucleic acid locus any of the non-naturalsystems disclosed herein, including an enzyme and at least one syntheticguide RNA (sgRNA) disclosed herein. The enzyme may form a complex withthe at least one sgRNA, and upon binding of the complex to the targetnucleic acid locus, may modify the target nucleic acid locus. Deliveringthe enzyme to said locus may comprise transfecting a cell with thesystem or nucleic acids encoding the system. Delivering the nuclease tosaid locus may comprise electroporating a cell with the system ornucleic acids encoding the system. Delivering the nuclease to said locusmay comprise incubating the system in a buffer with a nucleic acidcomprising the locus of interest. In some cases, the target nucleic acidlocus comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).The target nucleic acid locus may comprise genomic DNA, viral DNA, viralRNA, or bacterial DNA. The target nucleic acid locus may be within acell. The target nucleic acid locus may be in vitro. The target nucleicacid locus may be within a eukaryotic cell or a prokaryotic cell. Thecell may be an animal cell, a human cell, bacterial cell, archaeal cell,or a plant cell. The enzyme may induce a single or double-stranded breakat or proximal to the target locus of interest.

In cases where the target nucleic acid locus may be within a cell, theenzyme may be supplied as a nucleic acid containing an open readingframe encoding the enzyme having a RuvC_III domain having at least about75% (e.g., at least about 90%, at least about 91%, at least about 92%,at least about 93%, at least about 94%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, at least about 99%)identity to any one of SEQ ID NOs: 1827-2140. The deoxyribonucleic acid(DNA) containing an open reading frame encoding said endonuclease maycomprise a sequence substantially identical to any of SEQ ID NOs:5572-5575 or at variant having at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 55%, at least about 60%, at least about 65%, at least about 70%,at least about 75%, at least about 80%, at least about 85%, at leastabout 90%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, or at least about 99% identity to any one of SEQ IDNOs: 5572-5575. In some cases, the nucleic acid comprises a promoter towhich the open reading frame encoding the endonuclease is operablylinked. The promoter may be a CMV, EF1a, SV40, PGK1, Ubc, human betaactin, CAG, TRE, or CaMKIIa promoter. The endonuclease may be suppliedas a capped mRNA containing said open reading frame encoding saidendonuclease. The endonuclease may be supplied as a translatedpolypeptide. The at least one engineered sgRNA may be supplied asdeoxyribonucleic acid (DNA) containing a gene sequence encoding said atleast one engineered sgRNA operably linked to a ribonucleic acid (RNA)pol III promoter. In some cases, the organism may be eukaryotic. In somecases, the organism may be fungal. In some cases, the organism may behuman.

In some cases, the present disclosure may provide for an expressioncassette comprising the system disclosed herein, or the nucleic aciddescribed herein. In some cases, the expression cassette or nucleic acidmay be supplied as a vector. In some cases, the expression cassette,nucleic acid, or vector may be supplied in a cell. In some cases, thecell is a cell of a bacterium with a 16S rRNA gene having at least about90% (e.g., at least about 99%) identity to any one of SEQ ID NOs:5592-5595.

MG2 Enzymes

In one aspect, the present disclosure provides for an engineerednuclease system comprising (a) an endonuclease. In some cases, theendonuclease is a Cas endonuclease. In some cases, the endonuclease is aType II, Class II Cas endonuclease. The endonuclease may comprise aRuvC_III domain, wherein said RuvC_III domain has at least about 70%sequence identity to any one of SEQ ID NOs: 2141-2241. In some cases,the endonuclease may comprise a RuvC_III domain, wherein the RuvC_IIIdomain has at least about 20%, at least about 25%, at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 91%, at least about 92%,at least about 93%, at least about 94%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, at least about 99%identity to any one of SEQ ID NOs: 2141-2241. In some cases, theendonuclease may comprise a RuvC_III domain, wherein the substantiallyidentical to any one of SEQ ID NOs: 2141-2142. The endonuclease maycomprise a RuvC_III domain having at least about 70% sequence identityto any one of SEQ ID NOs: 2141-2142. In some cases, the endonuclease maycomprise a RuvC_III domain having at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about91%, at least about 92%, at least about 93%, at least about 94%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, at least about 99% identity to any one of SEQ ID NOs: 2141-2142. Insome cases, the endonuclease may comprise a RuvC_III domainsubstantially identical to any one of SEQ ID NOs: 2141-2142.

The endonuclease may comprise an HNH domain having at least about 70%identity to any one of SEQ ID NOs: 3955-4055. In some cases, theendonuclease may comprise an HNH domain having at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% identical to any one ofSEQ ID NOs: 3955-4055. The endonuclease may comprise an HNH domainsubstantially identical to any one of SEQ ID NOs: 3955-4055. Theendonuclease may comprise an HNH domain having at least about 70%identity to any one of SEQ ID NOs: 3955-3956. In some cases, theendonuclease may comprise an HNH domain having at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% identical to any one ofSEQ ID NOs: 3955-3956. The endonuclease may comprise an HNH domainsubstantially identical to any one of SEQ ID NOs: 3955-3956.

In some cases, the endonuclease may comprise a variant having at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 55%, at least about 60%, at leastabout 65%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, or at least about 99%identity to any one of SEQ ID NOs: 320-420. In some cases, theendonuclease may be substantially identical to any one of SEQ ID NOs:320-420. In some cases, the endonuclease may comprise a variant havingat least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, or at leastabout 99% identity to any one of SEQ ID NOs:320-321. In some cases, theendonuclease may be substantially identical to any one of SEQ ID NOs:320-321.

In some cases, the endonuclease may comprise a variant having one ormore nuclear localization sequences (NLSs). The NLS may be proximal tothe N- or C-terminus of said endonuclease. The NLS may be appendedN-terminal or C-terminal to any one of SEQ ID NOs: 320-420, or to avariant having at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 55%, atleast about 60%, at least about 65%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99% identity to any one of SEQ ID NOs: 320-420.The NLS may be an SV40 large T antigen NLS. The NLS may be a c-myc NLS.The NLS can comprise a sequence with at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 99% identityto any one of SEQ ID NOs: 5593-5608. The NLS can comprise a sequencesubstantially identical to any one of SEQ ID NOs: 5593-5608. The NLS cancomprise any of the sequences in Table 1 or a combination thereof.

In some cases, sequence identity may be determined by the BLASTP,CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology searchalgorithm. The sequence identity may be determined by the BLASTPalgorithm using parameters of a wordlength (W) of 3, an expectation (E)of 10, and using a BLOSUM62 scoring matrix setting gap costs atexistence of 11, extension of 1, and using a conditional compositionalscore matrix adjustment.

In some cases, the system above may comprise (b) at least one engineeredsynthetic guide ribonucleic acid (sgRNA) capable of forming a complexwith the endonuclease bearing a 5′ targeting region complementary to adesired cleavage sequence. In some cases, the 5′ targeting region maycomprises a PAM sequence compatible with the endonuclease. In somecases, the 5′ most nucleotide of the targeting region may be G. In somecases, the 5′ targeting region may be 15-23 nucleotides in length. Theguide sequence and the tracr sequence may be supplied as separateribonucleic acids (RNAs) or a single ribonucleic acid (RNA). The guideRNA may comprise a crRNA tracrRNA binding sequence 3′ to the targetingregion. The guide RNA may comprise a tracrRNA sequence preceded by a4-nucleotide linker 3′ to the crRNA tracrRNA binding region. The sgRNAmay comprise, from 5′ to 3′: a non-natural guide nucleic acid sequencecapable of hybridizing to a target sequence in a cell; and a tracrsequence. In some cases, the non-natural guide nucleic acid sequence andthe tracr sequence are covalently linked.

In some cases, the tracr sequence may have a particular sequence. Thetracr sequence may have at least about 80% to at least about 60-100(e.g., at least about 60, at least about 65, at least about 70, at leastabout 75, at least about 80, at least about 85, or at least about 90)consecutive nucleotides of a natural tracrRNA sequence. The tracrsequence may have at least about 80% sequence identity to at least about60-100 (e.g., at least about 60, at least about 65, at least about 70,at least about 75, at least about 80, at least about 85, or at leastabout 90) consecutive nucleotides of any one of SEQ ID NOs: 5490-5494.In some cases, the tracrRNA may have at least about 80%, at least about85%, at least about 90%, at least about 91%, at least about 92%, atleast about 93%, at least about 94%, at least about 95%, at least about96%, at least about 97%, at least about 98%, or at least about 99%identity to at least about 60-90 (e.g., at least about 60, at leastabout 65, at least about 70, at least about 75, at least about 80, atleast about 85, or at least about 90) consecutive nucleotides of any oneof SEQ ID NOs: 5490-5494. In some cases, the tracrRNA may besubstantially identical to at least about 60-100 (e.g., at least about60, at least about 65, at least about 70, at least about 75, at leastabout 80, at least about 85, or at least about 90) consecutivenucleotides of any one of SEQ ID NOs: 5490-5494. The tracrRNA maycomprise any of SEQ ID NOs: 5490-5494.

In some cases, the at least one engineered synthetic guide ribonucleicacid (sgRNA) capable of forming a complex with the endonuclease maycomprise a sequence having at least about 80% identity to SEQ ID NO:5465. The sgRNA may comprise a sequence having at least about 80%, atleast about 85%, at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, or at leastabout 99% identity to SEQ ID NO: 5465. The sgRNA may comprise a sequencesubstantially identical to SEQ ID NO: 5465.

In some cases, the system above may comprise two different sgRNAstargeting a first region and a second region for cleavage in a targetDNA locus, wherein the second region is 3′ to the first region. In somecases, the system above may comprise a single- or double-stranded DNArepair template comprising from 5′ to 3′: a first homology armcomprising a sequence of at least about 20 (e.g., at least about 40, 80,120, 150, 200, 300, 500, or 1 kb) nucleotides 5′ to the first region, asynthetic DNA sequence of at least about 10 nucleotides, and a secondhomology arm comprising a sequence of at least about 20 (e.g., at leastabout 40, 80, 120, 150, 200, 300, 500, or 1 kb) nucleotides 3′ to thesecond region.

In another aspect, the present disclosure provides a method formodifying a target nucleic acid locus of interest. The method maycomprise delivering to the target nucleic acid locus any of thenon-natural systems disclosed herein, including an enzyme and at leastone synthetic guide RNA (sgRNA) disclosed herein. The enzyme may form acomplex with the at least one sgRNA, and upon binding of the complex tothe target nucleic acid locus of interest, may modify the target nucleicacid locus of interest. Delivering the enzyme to said locus may comprisetransfecting a cell with the system or nucleic acids encoding thesystem. Delivering the nuclease to said locus may compriseelectroporating a cell with the system or nucleic acids encoding thesystem. Delivering the nuclease to said locus may comprise incubatingthe system in a buffer with a nucleic acid comprising the locus ofinterest. In some cases, the target nucleic acid locus comprisesdeoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The targetnucleic acid locus may comprise genomic DNA, viral DNA, viral RNA, orbacterial DNA. The target nucleic acid locus may be within a cell. Thetarget nucleic acid locus may be in vitro. The target nucleic acid locusmay be within a eukaryotic cell or a prokaryotic cell. The cell may bean animal cell, a human cell, bacterial cell, archaeal cell, or a plantcell. The enzyme may induce a single or double-stranded break at orproximal to the target locus of interest.

In cases where the target nucleic acid locus may be within a cell, theenzyme may be supplied as a nucleic acid containing an open readingframe encoding the enzyme having a RuvC_III domain having at least about75% (e.g., at least about 90%, at least about 91%, at least about 92%,at least about 93%, at least about 94%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, at least about 99%)identity to any one of SEQ ID NOs: 2141-2241. The deoxyribonucleic acid(DNA) containing an open reading frame encoding said endonuclease maycomprise a sequence substantially identical to any of SEQ ID NOs:5576-5577 or at variant having at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 55%, at least about 60%, at least about 65%, at least about 70%,at least about 75%, at least about 80%, at least about 85%, at leastabout 90%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, or at least about 99% identity to any one of SEQ IDNOs: 5576-5577. In some cases, the nucleic acid comprises a promoter towhich the open reading frame encoding the endonuclease is operablylinked. The promoter may be a CMV, EF1a, SV40, PGK1, Ubc, human betaactin, CAG, TRE, or CaMKIIa promoter. The endonuclease may be suppliedas a capped mRNA containing said open reading frame encoding saidendonuclease. The endonuclease may be supplied as a translatedpolypeptide. The at least one engineered sgRNA may be supplied asdeoxyribonucleic acid (DNA) containing a gene sequence encoding said atleast one engineered sgRNA operably linked to a ribonucleic acid (RNA)pol III promoter. In some cases, the organism may be eukaryotic. In somecases, the organism may be fungal. In some cases, the organism may behuman.

MG3 Enzymes

In one aspect, the present disclosure provides for an engineerednuclease system comprising (a) an endonuclease. In some cases, theendonuclease is a Cas endonuclease. In some cases, the endonuclease is aType II, Class II Cas endonuclease. The endonuclease may comprise aRuvC_III domain, wherein said RuvC_III domain has at least about 70%sequence identity to any one of SEQ ID NOs: 2242-2251. In some cases,the endonuclease may comprise a RuvC_III domain, wherein the RuvC_IIIdomain has at least about 20%, at least about 25%, at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 91%, at least about 92%,at least about 93%, at least about 94%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, at least about 99%identity to any one of SEQ ID NOs: 2242-2251. In some cases, theendonuclease may comprise a RuvC_III domain, wherein the substantiallyidentical to any one of SEQ ID NOs: 2242-2251. The endonuclease maycomprise a RuvC_III domain having at least about 70% sequence identityto any one of SEQ ID NOs: 2242-2244. In some cases, the endonuclease maycomprise a RuvC_III domain having at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about91%, at least about 92%, at least about 93%, at least about 94%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, at least about 99% identity to any one of SEQ ID NOs: 2242-2244. Insome cases, the endonuclease may comprise a RuvC_III domainsubstantially identical to any one of SEQ ID NOs: 2242-2244.

The endonuclease may comprise an HNH domain having at least about 70%identity to any one of SEQ ID NOs: 4056-4066. In some cases, theendonuclease may comprise an HNH domain having at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% identical to any one ofSEQ ID NOs: 4056-4066. The endonuclease may comprise an HNH domainsubstantially identical to any one of SEQ ID NOs: 4056-4066. Theendonuclease may comprise an HNH domain having at least about 70%identity to any one of SEQ ID NOs: 4056-4058. In some cases, theendonuclease may comprise an HNH domain having at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% identical to any one ofSEQ ID NOs: 4056-4058. The endonuclease may comprise an HNH domainsubstantially identical to any one of SEQ ID NOs: 4056-4058.

In some cases, the endonuclease may comprise a variant having at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 55%, at least about 60%, at leastabout 65%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, or at least about 99%identity to any one of SEQ ID NOs: 421-431. In some cases, theendonuclease may be substantially identical to any one of SEQ ID NOs:421-431. In some cases, the endonuclease may comprise a variant havingat least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, or at leastabout 99% identity to any one of SEQ ID NOs:421-423. In some cases, theendonuclease may be substantially identical to any one of SEQ ID NOs:421-423.

In some cases, the endonuclease may comprise a variant having one ormore nuclear localization sequences (NLSs). The NLS may be proximal tothe N- or C-terminus of said endonuclease. The NLS may be appendedN-terminal or C-terminal to any one of SEQ ID NOs: 421-431, or to avariant having at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 55%, atleast about 60%, at least about 65%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99% identity to any one of SEQ ID NOs: 421-431.The NLS may be an SV40 large T antigen NLS. The NLS may be a c-myc NLS.The NLS can comprise a sequence with at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 99% identityto any one of SEQ ID NOs: 5593-5608. The NLS can comprise a sequencesubstantially identical to any one of SEQ ID NOs: 5593-5608. The NLS cancomprise any of the sequences in Table 1 or a combination thereof.

In some cases, sequence identity may be determined by the BLASTP,CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology searchalgorithm. The sequence identity may be determined by the BLASTPalgorithm using parameters of a wordlength (W) of 3, an expectation (E)of 10, and using a BLOSUM62 scoring matrix setting gap costs atexistence of 11, extension of 1, and using a conditional compositionalscore matrix adjustment.

In some cases, the system above may comprise (b) at least one engineeredsynthetic guide ribonucleic acid (sgRNA) capable of forming a complexwith the endonuclease bearing a 5′ targeting region complementary to adesired cleavage sequence. In some cases, the 5′ targeting region maycomprises a PAM sequence compatible with the endonuclease. In somecases, the 5′ most nucleotide of the targeting region may be G. In somecases, the 5′ targeting region may be 15-23 nucleotides in length. Theguide sequence and the tracr sequence may be supplied as separateribonucleic acids (RNAs) or a single ribonucleic acid (RNA). The guideRNA may comprise a crRNA tracrRNA binding sequence 3′ to the targetingregion. The guide RNA may comprise a tracrRNA sequence preceded by a4-nucleotide linker 3′ to the crRNA tracrRNA binding region. The sgRNAmay comprise, from 5′ to 3′: a non-natural guide nucleic acid sequencecapable of hybridizing to a target sequence in a cell; and a tracrsequence. In some cases, the non-natural guide nucleic acid sequence andthe tracr sequence are covalently linked.

In some cases, the tracr sequence may have a particular sequence. Thetracr sequence may have at least about 80% to at least about 60-100(e.g., at least about 60, at least about 65, at least about 70, at leastabout 75, at least about 80, at least about 85, or at least about 90)consecutive nucleotides of a natural tracrRNA sequence. The tracrsequence may have at least about 80% sequence identity to at least about60-100 (e.g., at least about 60, at least about 65, at least about 70,at least about 75, at least about 80, at least about 85, or at leastabout 90) consecutive nucleotides of any one of SEQ ID NOs: 5495-5502.In some cases, the tracrRNA may have at least about 80%, at least about85%, at least about 90%, at least about 91%, at least about 92%, atleast about 93%, at least about 94%, at least about 95%, at least about96%, at least about 97%, at least about 98%, or at least about 99%identity to at least about 60-90 (e.g., at least about 60, at leastabout 65, at least about 70, at least about 75, at least about 80, atleast about 85, or at least about 90) consecutive nucleotides of any oneof SEQ ID NOs: 5495-5502. In some cases, the tracrRNA may besubstantially identical to at least about 60-100 (e.g., at least about60, at least about 65, at least about 70, at least about 75, at leastabout 80, at least about 85, or at least about 90) consecutivenucleotides of any one of SEQ ID NOs: 5495-5502. The tracrRNA maycomprise any of SEQ ID NOs: 5495-5502.

In some cases, the at least one engineered synthetic guide ribonucleicacid (sgRNA) capable of forming a complex with the endonuclease maycomprise a sequence having at least about 80% identity to any one of SEQID NOs: 5466-5467. The sgRNA may comprise a sequence having at leastabout 80%, at least about 85%, at least about 90%, at least about 91%,at least about 92%, at least about 93%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99% identity to any one of SEQ ID NOs: 5466-5467. ThesgRNA may comprise a sequence substantially identical to any one of SEQID NOs: 5466-5467.

In some cases, the system above may comprise two different sgRNAstargeting a first region and a second region for cleavage in a targetDNA locus, wherein the second region is 3′ to the first region. In somecases, the system above may comprise a single- or double-stranded DNArepair template comprising from 5′ to 3′: a first homology armcomprising a sequence of at least about 20 (e.g., at least about 40, 80,120, 150, 200, 300, 500, or 1 kb) nucleotides 5′ to the first region, asynthetic DNA sequence of at least about 10 nucleotides, and a secondhomology arm comprising a sequence of at least about 20 (e.g., at leastabout 40, 80, 120, 150, 200, 300, 500, or 1 kb) nucleotides 3′ to thesecond region.

In another aspect, the present disclosure provides a method formodifying a target nucleic acid locus of interest. The method maycomprise delivering to the target nucleic acid locus any of thenon-natural systems disclosed herein, including an enzyme and at leastone synthetic guide RNA (sgRNA) disclosed herein. The enzyme may form acomplex with the at least one sgRNA, and upon binding of the complex tothe target nucleic acid locus of interest, may modify the target nucleicacid locus of interest. Delivering the enzyme to said locus may comprisetransfecting a cell with the system or nucleic acids encoding thesystem. Delivering the nuclease to said locus may compriseelectroporating a cell with the system or nucleic acids encoding thesystem. Delivering the nuclease to said locus may comprise incubatingthe system in a buffer with a nucleic acid comprising the locus ofinterest. In some cases, the target nucleic acid locus comprisesdeoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The targetnucleic acid locus may comprise genomic DNA, viral DNA, viral RNA, orbacterial DNA. The target nucleic acid locus may be within a cell. Thetarget nucleic acid locus may be in vitro. The target nucleic acid locusmay be within a eukaryotic cell or a prokaryotic cell. The cell may bean animal cell, a human cell, bacterial cell, archaeal cell, or a plantcell. The enzyme may induce a single or double-stranded break at orproximal to the target locus of interest.

In cases where the target nucleic acid locus may be within a cell, theenzyme may be supplied as a nucleic acid containing an open readingframe encoding the enzyme having a RuvC_III domain having at least about75% (e.g., at least about 90%, at least about 91%, at least about 92%,at least about 93%, at least about 94%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, at least about 99%)identity to any one of SEQ ID NOs: 2242-2251. The deoxyribonucleic acid(DNA) containing an open reading frame encoding said endonuclease maycomprise a sequence substantially identical to any of SEQ ID NOs:5578-5580 or at variant having at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 55%, at least about 60%, at least about 65%, at least about 70%,at least about 75%, at least about 80%, at least about 85%, at leastabout 90%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, or at least about 99% identity to any one of SEQ IDNOs: 5578-5580. In some cases, the nucleic acid comprises a promoter towhich the open reading frame encoding the endonuclease is operablylinked. The promoter may be a CMV, EF1a, SV40, PGK1, Ubc, human betaactin, CAG, TRE, or CaMKIIa promoter. The endonuclease may be suppliedas a capped mRNA containing said open reading frame encoding saidendonuclease. The endonuclease may be supplied as a translatedpolypeptide. The at least one engineered sgRNA may be supplied asdeoxyribonucleic acid (DNA) containing a gene sequence encoding said atleast one engineered sgRNA operably linked to a ribonucleic acid (RNA)pol III promoter. In some cases, the organism may be eukaryotic. In somecases, the organism may be fungal. In some cases, the organism may behuman.

MG4 Enzymes

In one aspect, the present disclosure provides for an engineerednuclease system comprising (a) an endonuclease. In some cases, theendonuclease is a Cas endonuclease. In some cases, the endonuclease is aType II, Class II Cas endonuclease. The endonuclease may comprise aRuvC_III domain, wherein said RuvC_III domain has at least about 70%sequence identity to any one of SEQ ID NOs: 2253-2481. In some cases,the endonuclease may comprise a RuvC_III domain, wherein the RuvC_IIIdomain has at least about 20%, at least about 25%, at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 91%, at least about 92%,at least about 93%, at least about 94%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, at least about 99%identity to any one of SEQ ID NOs: 2253-2481. In some cases, theendonuclease may comprise a RuvC_III domain, wherein the substantiallyidentical to any one of SEQ ID NOs: 2253-2481. The endonuclease maycomprise a RuvC_III domain having at least about 70% sequence identityto any one of SEQ ID NOs: 2253-2481. In some cases, the endonuclease maycomprise a RuvC_III domain having at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about91%, at least about 92%, at least about 93%, at least about 94%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, at least about 99% identity to any one of SEQ ID NOs: 2253-2481. Insome cases, the endonuclease may comprise a RuvC_III domainsubstantially identical to any one of SEQ ID NOs: 2253-2481.

The endonuclease may comprise an HNH domain having at least about 70%identity to any one of SEQ ID NOs: 4067-4295. In some cases, theendonuclease may comprise an HNH domain having at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% identical to any one ofSEQ ID NOs: 4067-4295. The endonuclease may comprise an HNH domainsubstantially identical to any one of SEQ ID NOs: 4067-4295. Theendonuclease may comprise an HNH domain having at least about 70%identity to any one of SEQ ID NOs: 4067-4295. In some cases, theendonuclease may comprise an HNH domain having at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% identical to any one ofSEQ ID NOs: 4067-4295. The endonuclease may comprise an HNH domainsubstantially identical to any one of SEQ ID NOs: 4067-4295.

In some cases, the endonuclease may comprise a variant having at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 55%, at least about 60%, at leastabout 65%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, or at least about 99%identity to any one of SEQ ID NOs: 432-660. In some cases, theendonuclease may be substantially identical to any one of SEQ ID NOs:432-660. In some cases, the endonuclease may comprise a variant havingat least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, or at leastabout 99% identity to any one of SEQ ID NOs: 432-660. In some cases, theendonuclease may be substantially identical to any one of SEQ ID NOs:432-660.

In some cases, the endonuclease may comprise a variant having one ormore nuclear localization sequences (NLSs). The NLS may be proximal tothe N- or C-terminus of said endonuclease. The NLS may be appendedN-terminal or C-terminal to any one of SEQ ID NOs: 432-660, or to avariant having at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 55%, atleast about 60%, at least about 65%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99% identity to any one of SEQ ID NOs: 432-660.The NLS may be an SV40 large T antigen NLS. The NLS may be a c-myc NLS.The NLS can comprise a sequence with at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 99% identityto any one of SEQ ID NOs: 5593-5608. The NLS can comprise a sequencesubstantially identical to any one of SEQ ID NOs: 5593-5608. The NLS cancomprise any of the sequences in Table 1 or a combination thereof.

In some cases, sequence identity may be determined by the BLASTP,CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology searchalgorithm. The sequence identity may be determined by the BLASTPalgorithm using parameters of a wordlength (W) of 3, an expectation (E)of 10, and using a BLOSUM62 scoring matrix setting gap costs atexistence of 11, extension of 1, and using a conditional compositionalscore matrix adjustment.

In some cases, the system above may comprise (b) at least one engineeredsynthetic guide ribonucleic acid (sgRNA) capable of forming a complexwith the endonuclease bearing a 5′ targeting region complementary to adesired cleavage sequence. In some cases, the 5′ targeting region maycomprises a PAM sequence compatible with the endonuclease. In somecases, the 5′ most nucleotide of the targeting region may be G. In somecases, the 5′ targeting region may be 15-23 nucleotides in length. Theguide sequence and the tracr sequence may be supplied as separateribonucleic acids (RNAs) or a single ribonucleic acid (RNA). The guideRNA may comprise a crRNA tracrRNA binding sequence 3′ to the targetingregion. The guide RNA may comprise a tracrRNA sequence preceded by a4-nucleotide linker 3′ to the crRNA tracrRNA binding region. The sgRNAmay comprise, from 5′ to 3′: a non-natural guide nucleic acid sequencecapable of hybridizing to a target sequence in a cell; and a tracrsequence. In some cases, the non-natural guide nucleic acid sequence andthe tracr sequence are covalently linked.

In some cases, the tracr sequence may have a particular sequence. Thetracr sequence may have at least about 80% to at least about 60-100(e.g., at least about 60, at least about 65, at least about 70, at leastabout 75, at least about 80, at least about 85, or at least about 90)consecutive nucleotides of a natural tracrRNA sequence. The tracrsequence may have at least about 80% sequence identity to at least about60-100 (e.g., at least about 60, at least about 65, at least about 70,at least about 75, at least about 80, at least about 85, or at leastabout 90) consecutive nucleotides of SEQ ID NO: 5503. In some cases, thetracrRNA may have at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% identity to at leastabout 60-90 (e.g., at least about 60, at least about 65, at least about70, at least about 75, at least about 80, at least about 85, or at leastabout 90) consecutive nucleotides of SEQ ID NO: 5503. In some cases, thetracrRNA may be substantially identical to at least about 60-100 (e.g.,at least about 60, at least about 65, at least about 70, at least about75, at least about 80, at least about 85, or at least about 90)consecutive nucleotides of SEQ ID NO: 5503. The tracrRNA may compriseSEQ ID NO: 5503.

In some cases, the at least one engineered synthetic guide ribonucleicacid (sgRNA) capable of forming a complex with the endonuclease maycomprise a sequence having at least about 80% identity to SEQ ID NO:5468. The sgRNA may comprise a sequence having at least about 80%, atleast about 85%, at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, or at leastabout 99% identity to SEQ ID NO: 5468. The sgRNA may comprise a sequencesubstantially identical to SEQ ID NO: 5468.

In some cases, the system above may comprise two different sgRNAstargeting a first region and a second region for cleavage in a targetDNA locus, wherein the second region is 3′ to the first region. In somecases, the system above may comprise a single- or double-stranded DNArepair template comprising from 5′ to 3′: a first homology armcomprising a sequence of at least about 20 (e.g., at least about 40, 80,120, 150, 200, 300, 500, or 1 kb) nucleotides 5′ to the first region, asynthetic DNA sequence of at least about 10 nucleotides, and a secondhomology arm comprising a sequence of at least about 20 (e.g., at leastabout 40, 80, 120, 150, 200, 300, 500, or 1 kb) nucleotides 3′ to thesecond region.

In another aspect, the present disclosure provides a method formodifying a target nucleic acid locus of interest. The method maycomprise delivering to the target nucleic acid locus any of thenon-natural systems disclosed herein, including an enzyme and at leastone synthetic guide RNA (sgRNA) disclosed herein. The enzyme may form acomplex with the at least one sgRNA, and upon binding of the complex tothe target nucleic acid locus of interest, may modify the target nucleicacid locus of interest. Delivering the enzyme to said locus may comprisetransfecting a cell with the system or nucleic acids encoding thesystem. Delivering the nuclease to said locus may compriseelectroporating a cell with the system or nucleic acids encoding thesystem. Delivering the nuclease to said locus may comprise incubatingthe system in a buffer with a nucleic acid comprising the locus ofinterest. In some cases, the target nucleic acid locus comprisesdeoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The targetnucleic acid locus may comprise genomic DNA, viral DNA, viral RNA, orbacterial DNA. The target nucleic acid locus may be within a cell. Thetarget nucleic acid locus may be in vitro. The target nucleic acid locusmay be within a eukaryotic cell or a prokaryotic cell. The cell may bean animal cell, a human cell, bacterial cell, archaeal cell, or a plantcell. The enzyme may induce a single or double-stranded break at orproximal to the target locus of interest.

In cases where the target nucleic acid locus may be within a cell, theenzyme may be supplied as a nucleic acid containing an open readingframe encoding the enzyme having a RuvC_III domain having at least about75% (e.g., at least about 90%, at least about 91%, at least about 92%,at least about 93%, at least about 94%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, at least about 99%)identity to any one of SEQ ID NOs: 2253-2481. In some cases, the nucleicacid comprises a promoter to which the open reading frame encoding theendonuclease is operably linked. The promoter may be a CMV, EF1a, SV40,PGK1, Ubc, human beta actin, CAG, TRE, or CaMKIIa promoter. Theendonuclease may be supplied as a capped mRNA containing said openreading frame encoding said endonuclease. The endonuclease may besupplied as a translated polypeptide. The at least one engineered sgRNAmay be supplied as deoxyribonucleic acid (DNA) containing a genesequence encoding said at least one engineered sgRNA operably linked toa ribonucleic acid (RNA) pol III promoter. In some cases, the organismmay be eukaryotic. In some cases, the organism may be fungal. In somecases, the organism may be human.

MG6 Enzymes

In one aspect, the present disclosure provides for an engineerednuclease system comprising (a) an endonuclease. In some cases, theendonuclease is a Cas endonuclease. In some cases, the endonuclease is aType II, Class II Cas endonuclease. The endonuclease may comprise aRuvC_III domain, wherein said RuvC_III domain has at least about 70%sequence identity to any one of SEQ ID NOs: 2482-2489. In some cases,the endonuclease may comprise a RuvC_III domain, wherein the RuvC_IIIdomain has at least about 20%, at least about 25%, at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 91%, at least about 92%,at least about 93%, at least about 94%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, at least about 99%identity to any one of SEQ ID NOs: 2482-2489. In some cases, theendonuclease may comprise a RuvC_III domain, wherein the substantiallyidentical to any one of SEQ ID NOs: 2482-2489.

The endonuclease may comprise an HNH domain having at least about 70%identity to any one of SEQ ID NOs: 4296-4303. In some cases, theendonuclease may comprise an HNH domain having at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% identical to any one ofSEQ ID NOs: 4296-4303. The endonuclease may comprise an HNH domainsubstantially identical to any one of SEQ ID NOs: 4056-4066.

In some cases, the endonuclease may comprise a variant having at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 55%, at least about 60%, at leastabout 65%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, or at least about 99%identity to any one of SEQ ID NOs: 661-668. In some cases, theendonuclease may be substantially identical to any one of SEQ ID NOs:661-668.

In some cases, the endonuclease may comprise a variant having one ormore nuclear localization sequences (NLSs). The NLS may be proximal tothe N- or C-terminus of said endonuclease. The NLS may be appendedN-terminal or C-terminal to any one of SEQ ID NOs: 661-668, or to avariant having at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 55%, atleast about 60%, at least about 65%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99% identity to any one of SEQ ID NOs: 661-668.The NLS may be an SV40 large T antigen NLS. The NLS may be a c-myc NLS.The NLS can comprise a sequence with at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 99% identityto any one of SEQ ID NOs: 5593-5608. The NLS can comprise a sequencesubstantially identical to any one of SEQ ID NOs: 5593-5608. The NLS cancomprise any of the sequences in Table 1 or a combination thereof.

In some cases, sequence identity may be determined by the BLASTP,CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology searchalgorithm. The sequence identity may be determined by the BLASTPalgorithm using parameters of a wordlength (W) of 3, an expectation (E)of 10, and using a BLOSUM62 scoring matrix setting gap costs atexistence of 11, extension of 1, and using a conditional compositionalscore matrix adjustment.

In some cases, the system above may comprise (b) at least one engineeredsynthetic guide ribonucleic acid (sgRNA) capable of forming a complexwith the endonuclease bearing a 5′ targeting region complementary to adesired cleavage sequence. In some cases, the 5′ targeting region maycomprises a PAM sequence compatible with the endonuclease. In somecases, the 5′ most nucleotide of the targeting region may be G. In somecases, the 5′ targeting region may be 15-23 nucleotides in length. Theguide sequence and the tracr sequence may be supplied as separateribonucleic acids (RNAs) or a single ribonucleic acid (RNA). The guideRNA may comprise a crRNA tracrRNA binding sequence 3′ to the targetingregion. The guide RNA may comprise a tracrRNA sequence preceded by a4-nucleotide linker 3′ to the crRNA tracrRNA binding region. The sgRNAmay comprise, from 5′ to 3′: a non-natural guide nucleic acid sequencecapable of hybridizing to a target sequence in a cell; and a tracrsequence. In some cases, the non-natural guide nucleic acid sequence andthe tracr sequence are covalently linked.

In some cases, the tracr sequence may have a particular sequence. Thetracr sequence may have at least about 80% to at least about 60-100(e.g., at least about 60, at least about 65, at least about 70, at leastabout 75, at least about 80, at least about 85, or at least about 90)consecutive nucleotides of a natural tracrRNA sequence.

In some cases, the system above may comprise two different guide RNAstargeting a first region and a second region for cleavage in a targetDNA locus, wherein the second region is 3′ to the first region. In somecases, the system above may comprise a single- or double-stranded DNArepair template comprising from 5′ to 3′: a first homology armcomprising a sequence of at least about 20 (e.g., at least about 40, 80,120, 150, 200, 300, 500, or 1 kb) nucleotides 5′ to the first region, asynthetic DNA sequence of at least about 10 nucleotides, and a secondhomology arm comprising a sequence of at least about 20 (e.g., at leastabout 40, 80, 120, 150, 200, 300, 500, or 1 kb) nucleotides 3′ to thesecond region.

In another aspect, the present disclosure provides a method formodifying a target nucleic acid locus of interest. The method maycomprise delivering to the target nucleic acid locus any of thenon-natural systems disclosed herein, including an enzyme and at leastone synthetic guide RNA (sgRNA) disclosed herein. The enzyme may form acomplex with the at least one sgRNA, and upon binding of the complex tothe target nucleic acid locus of interest, may modify the target nucleicacid locus of interest. Delivering the enzyme to said locus may comprisetransfecting a cell with the system or nucleic acids encoding thesystem. Delivering the nuclease to said locus may compriseelectroporating a cell with the system or nucleic acids encoding thesystem. Delivering the nuclease to said locus may comprise incubatingthe system in a buffer with a nucleic acid comprising the locus ofinterest. In some cases, the target nucleic acid locus comprisesdeoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The targetnucleic acid locus may comprise genomic DNA, viral DNA, viral RNA, orbacterial DNA. The target nucleic acid locus may be within a cell. Thetarget nucleic acid locus may be in vitro. The target nucleic acid locusmay be within a eukaryotic cell or a prokaryotic cell. The cell may bean animal cell, a human cell, bacterial cell, archaeal cell, or a plantcell. The enzyme may induce a single or double-stranded break at orproximal to the target locus of interest.

In cases where the target nucleic acid locus may be within a cell, theenzyme may be supplied as a nucleic acid containing an open readingframe encoding the enzyme having a RuvC_III domain having at least about75% (e.g., at least about 90%, at least about 91%, at least about 92%,at least about 93%, at least about 94%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, at least about 99%)identity to any one of SEQ ID NOs: 2482-2489. In some cases, the nucleicacid comprises a promoter to which the open reading frame encoding theendonuclease is operably linked. The promoter may be a CMV, EF1a, SV40,PGK1, Ubc, human beta actin, CAG, TRE, or CaMKIIa promoter. Theendonuclease may be supplied as a capped mRNA containing said openreading frame encoding said endonuclease. The endonuclease may besupplied as a translated polypeptide. The at least one engineered sgRNAmay be supplied as deoxyribonucleic acid (DNA) containing a genesequence encoding said at least one engineered sgRNA operably linked toa ribonucleic acid (RNA) pol III promoter. In some cases, the organismmay be eukaryotic. In some cases, the organism may be fungal. In somecases, the organism may be human.

MG7 Enzymes

In one aspect, the present disclosure provides for an engineerednuclease system comprising (a) an endonuclease. In some cases, theendonuclease is a Cas endonuclease. In some cases, the endonuclease is aType II, Class II Cas endonuclease. The endonuclease may comprise aRuvC_III domain, wherein said RuvC_III domain has at least about 70%sequence identity to any one of SEQ ID NOs: 2490-2498. In some cases,the endonuclease may comprise a RuvC_III domain, wherein the RuvC_IIIdomain has at least about 20%, at least about 25%, at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 91%, at least about 92%,at least about 93%, at least about 94%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, at least about 99%identity to any one of SEQ ID NOs: 2490-2498. In some cases, theendonuclease may comprise a RuvC_III domain, wherein the substantiallyidentical to any one of SEQ ID NOs: 2490-2498. The endonuclease maycomprise a RuvC_III domain having at least about 70% sequence identityto any one of SEQ ID NOs: 2490-2498. In some cases, the endonuclease maycomprise a RuvC_III domain having at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about91%, at least about 92%, at least about 93%, at least about 94%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, at least about 99% identity to any one of SEQ ID NOs: 2490-2498. Insome cases, the endonuclease may comprise a RuvC_III domainsubstantially identical to any one of SEQ ID NOs: 2490-2498.

The endonuclease may comprise an HNH domain having at least about 70%identity to any one of SEQ ID NOs: 4304-4312. In some cases, theendonuclease may comprise an HNH domain having at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% identical to any one ofSEQ ID NOs: 4304-4312. The endonuclease may comprise an HNH domainsubstantially identical to any one of SEQ ID NOs: 4304-4312. Theendonuclease may comprise an HNH domain having at least about 70%identity to any one of SEQ ID NOs: 4304-4312. In some cases, theendonuclease may comprise an HNH domain having at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% identical to any one ofSEQ ID NOs: 4304-4312. The endonuclease may comprise an HNH domainsubstantially identical to any one of SEQ ID NOs: 4304-4312.

In some cases, the endonuclease may comprise a variant having at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 55%, at least about 60%, at leastabout 65%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, or at least about 99%identity to any one of SEQ ID NOs: 669-677. In some cases, theendonuclease may be substantially identical to any one of SEQ ID NOs:669-677. In some cases, the endonuclease may comprise a variant havingat least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, or at leastabout 99% identity to any one of SEQ ID NOs: 669-677. In some cases, theendonuclease may be substantially identical to any one of SEQ ID NOs:669-677.

In some cases, the endonuclease may comprise a variant having one ormore nuclear localization sequences (NLSs). The NLS may be proximal tothe N- or C-terminus of said endonuclease. The NLS may be appendedN-terminal or C-terminal to any one of SEQ ID NOs: 669-677, or to avariant having at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 55%, atleast about 60%, at least about 65%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99% identity to any one of SEQ ID NOs: 669-677.The NLS may be an SV40 large T antigen NLS. The NLS may be a c-myc NLS.The NLS can comprise a sequence with at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 99% identityto any one of SEQ ID NOs: 5593-5608. The NLS can comprise a sequencesubstantially identical to any one of SEQ ID NOs: 5593-5608. The NLS cancomprise any of the sequences in Table 1 or a combination thereof.

In some cases, sequence identity may be determined by the BLASTP,CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology searchalgorithm. The sequence identity may be determined by the BLASTPalgorithm using parameters of a wordlength (W) of 3, an expectation (E)of 10, and using a BLOSUM62 scoring matrix setting gap costs atexistence of 11, extension of 1, and using a conditional compositionalscore matrix adjustment.

In some cases, the system above may comprise (b) at least one engineeredsynthetic guide ribonucleic acid (sgRNA) capable of forming a complexwith the endonuclease bearing a 5′ targeting region complementary to adesired cleavage sequence. In some cases, the 5′ targeting region maycomprises a PAM sequence compatible with the endonuclease. In somecases, the 5′ most nucleotide of the targeting region may be G. In somecases, the 5′ targeting region may be 15-23 nucleotides in length. Theguide sequence and the tracr sequence may be supplied as separateribonucleic acids (RNAs) or a single ribonucleic acid (RNA). The guideRNA may comprise a crRNA tracrRNA binding sequence 3′ to the targetingregion. The guide RNA may comprise a tracrRNA sequence preceded by a4-nucleotide linker 3′ to the crRNA tracrRNA binding region. The sgRNAmay comprise, from 5′ to 3′: a non-natural guide nucleic acid sequencecapable of hybridizing to a target sequence in a cell; and a tracrsequence. In some cases, the non-natural guide nucleic acid sequence andthe tracr sequence are covalently linked.

In some cases, the tracr sequence may have a particular sequence. Thetracr sequence may have at least about 80% to at least about 60-100(e.g., at least about 60, at least about 65, at least about 70, at leastabout 75, at least about 80, at least about 85, or at least about 90)consecutive nucleotides of a natural tracrRNA sequence. The tracrsequence may have at least about 80% sequence identity to at least about60-100 (e.g., at least about 60, at least about 65, at least about 70,at least about 75, at least about 80, at least about 85, or at leastabout 90) consecutive nucleotides of SEQ ID NO: 5504. In some cases, thetracrRNA may have at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% identity to at leastabout 60-90 (e.g., at least about 60, at least about 65, at least about70, at least about 75, at least about 80, at least about 85, or at leastabout 90) consecutive nucleotides of SEQ ID NO: 5504. In some cases, thetracrRNA may be substantially identical to at least about 60-100 (e.g.,at least about 60, at least about 65, at least about 70, at least about75, at least about 80, at least about 85, or at least about 90)consecutive nucleotides of SEQ ID NO: 5504. The tracrRNA may compriseSEQ ID NO: 5504.

In some cases, the system above may comprise two different sgRNAstargeting a first region and a second region for cleavage in a targetDNA locus, wherein the second region is 3′ to the first region. In somecases, the system above may comprise a single- or double-stranded DNArepair template comprising from 5′ to 3′: a first homology armcomprising a sequence of at least about 20 (e.g., at least about 40, 80,120, 150, 200, 300, 500, or 1 kb) nucleotides 5′ to the first region, asynthetic DNA sequence of at least about 10 nucleotides, and a secondhomology arm comprising a sequence of at least about 20 (e.g., at leastabout 40, 80, 120, 150, 200, 300, 500, or 1 kb) nucleotides 3′ to thesecond region.

In another aspect, the present disclosure provides a method formodifying a target nucleic acid locus of interest. The method maycomprise delivering to the target nucleic acid locus any of thenon-natural systems disclosed herein, including an enzyme and at leastone synthetic guide RNA (sgRNA) disclosed herein. The enzyme may form acomplex with the at least one sgRNA, and upon binding of the complex tothe target nucleic acid locus of interest, may modify the target nucleicacid locus of interest. Delivering the enzyme to said locus may comprisetransfecting a cell with the system or nucleic acids encoding thesystem. Delivering the nuclease to said locus may compriseelectroporating a cell with the system or nucleic acids encoding thesystem. Delivering the nuclease to said locus may comprise incubatingthe system in a buffer with a nucleic acid comprising the locus ofinterest. In some cases, the target nucleic acid locus comprisesdeoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The targetnucleic acid locus may comprise genomic DNA, viral DNA, viral RNA, orbacterial DNA. The target nucleic acid locus may be within a cell. Thetarget nucleic acid locus may be in vitro. The target nucleic acid locusmay be within a eukaryotic cell or a prokaryotic cell. The cell may bean animal cell, a human cell, bacterial cell, archaeal cell, or a plantcell. The enzyme may induce a single or double-stranded break at orproximal to the target locus of interest.

In cases where the target nucleic acid locus may be within a cell, theenzyme may be supplied as a nucleic acid containing an open readingframe encoding the enzyme having a RuvC_III domain having at least about75% (e.g., at least about 90%, at least about 91%, at least about 92%,at least about 93%, at least about 94%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, at least about 99%)identity to any one of SEQ ID NOs: 2490-2498. In some cases, the nucleicacid comprises a promoter to which the open reading frame encoding theendonuclease is operably linked. The promoter may be a CMV, EF1a, SV40,PGK1, Ubc, human beta actin, CAG, TRE, or CaMKIIa promoter. Theendonuclease may be supplied as a capped mRNA containing said openreading frame encoding said endonuclease.

The endonuclease may be supplied as a translated polypeptide. The atleast one engineered sgRNA may be supplied as deoxyribonucleic acid(DNA) containing a gene sequence encoding said at least one engineeredsgRNA operably linked to a ribonucleic acid (RNA) pol III promoter. Insome cases, the organism may be eukaryotic. In some cases, the organismmay be fungal. In some cases, the organism may be human.

MG14 Enzymes

In one aspect, the present disclosure provides for an engineerednuclease system comprising (a) an endonuclease. In some cases, theendonuclease is a Cas endonuclease. In some cases, the endonuclease is aType II, Class II Cas endonuclease. The endonuclease may comprise aRuvC_III domain, wherein said RuvC_III domain has at least about 70%sequence identity to any one of SEQ ID NOs: 2499-2750. In some cases,the endonuclease may comprise a RuvC_III domain, wherein the RuvC_IIIdomain has at least about 20%, at least about 25%, at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 91%, at least about 92%,at least about 93%, at least about 94%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, at least about 99%identity to any one of SEQ ID NOs: 2499-2750. In some cases, theendonuclease may comprise a RuvC_III domain, wherein the substantiallyidentical to any one of SEQ ID NOs: 2499-2750. The endonuclease maycomprise a RuvC_III domain having at least about 70% sequence identityto any one of SEQ ID NOs: 2499-2750. In some cases, the endonuclease maycomprise a RuvC_III domain having at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about91%, at least about 92%, at least about 93%, at least about 94%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, at least about 99% identity to any one of SEQ ID NOs: 2499-2750. Insome cases, the endonuclease may comprise a RuvC_III domainsubstantially identical to any one of SEQ ID NOs: 2499-2750.

The endonuclease may comprise an HNH domain having at least about 70%identity to any one of SEQ ID NOs: 4313-4564. In some cases, theendonuclease may comprise an HNH domain having at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% identical to any one ofSEQ ID NOs: 4313-4564. The endonuclease may comprise an HNH domainsubstantially identical to any one of SEQ ID NOs: 4313-4564. Theendonuclease may comprise an HNH domain having at least about 70%identity to any one of SEQ ID NOs: 4313-4564. In some cases, theendonuclease may comprise an HNH domain having at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% identical to any one ofSEQ ID NOs: 4067-4295. The endonuclease may comprise an HNH domainsubstantially identical to any one of SEQ ID NOs: 4313-4564.

In some cases, the endonuclease may comprise a variant having at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 55%, at least about 60%, at leastabout 65%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, or at least about 99%identity to any one of SEQ ID NOs: 678-929. In some cases, theendonuclease may be substantially identical to any one of SEQ ID NOs:678-929. In some cases, the endonuclease may comprise a variant havingat least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, or at leastabout 99% identity to any one of SEQ ID NOs: 678-929. In some cases, theendonuclease may be substantially identical to any one of SEQ ID NOs:678-929.

In some cases, the endonuclease may comprise a variant having one ormore nuclear localization sequences (NLSs). The NLS may be proximal tothe N- or C-terminus of said endonuclease. The NLS may be appendedN-terminal or C-terminal to any one of SEQ ID NOs: 678-929, or to avariant having at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 55%, atleast about 60%, at least about 65%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99% identity to any one of SEQ ID NOs: 678-929.The NLS may be an SV40 large T antigen NLS. The NLS may be a c-myc NLS.The NLS can comprise a sequence with at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 99% identityto any one of SEQ ID NOs: 5593-5608. The NLS can comprise a sequencesubstantially identical to any one of SEQ ID NOs: 5593-5608. The NLS cancomprise any of the sequences in Table 1 or a combination thereof.

In some cases, sequence identity may be determined by the BLASTP,CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology searchalgorithm. The sequence identity may be determined by the BLASTPalgorithm using parameters of a wordlength (W) of 3, an expectation (E)of 10, and using a BLOSUM62 scoring matrix setting gap costs atexistence of 11, extension of 1, and using a conditional compositionalscore matrix adjustment.

In some cases, the system above may comprise (b) at least one engineeredsynthetic guide ribonucleic acid (sgRNA) capable of forming a complexwith the endonuclease bearing a 5′ targeting region complementary to adesired cleavage sequence. In some cases, the 5′ targeting region maycomprises a PAM sequence compatible with the endonuclease. In somecases, the 5′ most nucleotide of the targeting region may be G. In somecases, the 5′ targeting region may be 15-23 nucleotides in length. Theguide sequence and the tracr sequence may be supplied as separateribonucleic acids (RNAs) or a single ribonucleic acid (RNA). The guideRNA may comprise a crRNA tracrRNA binding sequence 3′ to the targetingregion. The guide RNA may comprise a tracrRNA sequence preceded by a4-nucleotide linker 3′ to the crRNA tracrRNA binding region. The sgRNAmay comprise, from 5′ to 3′: a non-natural guide nucleic acid sequencecapable of hybridizing to a target sequence in a cell; and a tracrsequence. In some cases, the non-natural guide nucleic acid sequence andthe tracr sequence are covalently linked.

In some cases, the tracr sequence may have a particular sequence. Thetracr sequence may have at least about 80% to at least about 60-100(e.g., at least about 60, at least about 65, at least about 70, at leastabout 75, at least about 80, at least about 85, or at least about 90)consecutive nucleotides of a natural tracrRNA sequence. The tracrsequence may have at least about 80% sequence identity to at least about60-100 (e.g., at least about 60, at least about 65, at least about 70,at least about 75, at least about 80, at least about 85, or at leastabout 90) consecutive nucleotides of SEQ ID NO: 5505. In some cases, thetracrRNA may have at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% identity to at leastabout 60-90 (e.g., at least about 60, at least about 65, at least about70, at least about 75, at least about 80, at least about 85, or at leastabout 90) consecutive nucleotides of SEQ ID NO: 5505. In some cases, thetracrRNA may be substantially identical to at least about 60-100 (e.g.,at least about 60, at least about 65, at least about 70, at least about75, at least about 80, at least about 85, or at least about 90)consecutive nucleotides of SEQ ID NO: 5505. The tracrRNA may compriseSEQ ID NO: 5505.

In some cases, the at least one engineered synthetic guide ribonucleicacid (sgRNA) capable of forming a complex with the endonuclease maycomprise a sequence having at least about 80% identity to SEQ ID NO:5469. The sgRNA may comprise a sequence having at least about 80%, atleast about 85%, at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, or at leastabout 99% identity to SEQ ID NO: 5469. The sgRNA may comprise a sequencesubstantially identical to SEQ ID NO: 5469.

In some cases, the system above may comprise two different sgRNAstargeting a first region and a second region for cleavage in a targetDNA locus, wherein the second region is 3′ to the first region. In somecases, the system above may comprise a single- or double-stranded DNArepair template comprising from 5′ to 3′: a first homology armcomprising a sequence of at least about 20 (e.g., at least about 40, 80,120, 150, 200, 300, 500, or 1 kb) nucleotides 5′ to the first region, asynthetic DNA sequence of at least about 10 nucleotides, and a secondhomology arm comprising a sequence of at least about 20 (e.g., at leastabout 40, 80, 120, 150, 200, 300, 500, or 1 kb) nucleotides 3′ to thesecond region.

In another aspect, the present disclosure provides a method formodifying a target nucleic acid locus of interest. The method maycomprise delivering to the target nucleic acid locus any of thenon-natural systems disclosed herein, including an enzyme and at leastone synthetic guide RNA (sgRNA) disclosed herein. The enzyme may form acomplex with the at least one sgRNA, and upon binding of the complex tothe target nucleic acid locus of interest, may modify the target nucleicacid locus of interest. Delivering the enzyme to said locus may comprisetransfecting a cell with the system or nucleic acids encoding thesystem. Delivering the nuclease to said locus may compriseelectroporating a cell with the system or nucleic acids encoding thesystem. Delivering the nuclease to said locus may comprise incubatingthe system in a buffer with a nucleic acid comprising the locus ofinterest. In some cases, the target nucleic acid locus comprisesdeoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The targetnucleic acid locus may comprise genomic DNA, viral DNA, viral RNA, orbacterial DNA. The target nucleic acid locus may be within a cell. Thetarget nucleic acid locus may be in vitro. The target nucleic acid locusmay be within a eukaryotic cell or a prokaryotic cell. The cell may bean animal cell, a human cell, bacterial cell, archaeal cell, or a plantcell. The enzyme may induce a single or double-stranded break at orproximal to the target locus of interest.

In cases where the target nucleic acid locus may be within a cell, theenzyme may be supplied as a nucleic acid containing an open readingframe encoding the enzyme having a RuvC_III domain having at least about75% (e.g., at least about 90%, at least about 91%, at least about 92%,at least about 93%, at least about 94%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, at least about 99%)identity to any one of SEQ ID NOs: 2499-2750. The deoxyribonucleic acid(DNA) containing an open reading frame encoding said endonuclease maycomprise a sequence substantially identical to SEQ ID NO: 5581 or atvariant having at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 55%, atleast about 60%, at least about 65%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99% identity to SEQ ID NO: 5581. In some cases,the nucleic acid comprises a promoter to which the open reading frameencoding the endonuclease is operably linked. The promoter may be a CMV,EF1a, SV40, PGK1, Ubc, human beta actin, CAG, TRE, or CaMKIIa promoter.The endonuclease may be supplied as a capped mRNA containing said openreading frame encoding said endonuclease. The endonuclease may besupplied as a translated polypeptide. The at least one engineered sgRNAmay be supplied as deoxyribonucleic acid (DNA) containing a genesequence encoding said at least one engineered sgRNA operably linked toa ribonucleic acid (RNA) pol III promoter. In some cases, the organismmay be eukaryotic. In some cases, the organism may be fungal. In somecases, the organism may be human.

MG15 Enzymes

In one aspect, the present disclosure provides for an engineerednuclease system comprising (a) an endonuclease. In some cases, theendonuclease is a Cas endonuclease. In some cases, the endonuclease is aType II, Class II Cas endonuclease. The endonuclease may comprise aRuvC_III domain, wherein said RuvC_III domain has at least about 70%sequence identity to any one of SEQ ID NOs: 2751-2913. In some cases,the endonuclease may comprise a RuvC_III domain, wherein the RuvC_IIIdomain has at least about 20%, at least about 25%, at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 91%, at least about 92%,at least about 93%, at least about 94%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, at least about 99%identity to any one of SEQ ID NOs: 2751-2913. In some cases, theendonuclease may comprise a RuvC_III domain, wherein the substantiallyidentical to any one of SEQ ID NOs: 2751-2913. The endonuclease maycomprise a RuvC_III domain having at least about 70% sequence identityto any one of SEQ ID NOs: 2751-2913. In some cases, the endonuclease maycomprise a RuvC_III domain having at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about91%, at least about 92%, at least about 93%, at least about 94%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, at least about 99% identity to any one of SEQ ID NOs: 2751-2913. Insome cases, the endonuclease may comprise a RuvC_III domainsubstantially identical to any one of SEQ ID NOs: 2751-2913.

The endonuclease may comprise an HNH domain having at least about 70%identity to any one of SEQ ID NOs: 4565-4727. In some cases, theendonuclease may comprise an HNH domain having at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% identical to any one ofSEQ ID NOs: 4565-4727. The endonuclease may comprise an HNH domainsubstantially identical to any one of SEQ ID NOs: 4565-4727. Theendonuclease may comprise an HNH domain having at least about 70%identity to any one of SEQ ID NOs: 4565-4727. In some cases, theendonuclease may comprise an HNH domain having at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% identical to any one ofSEQ ID NOs: 4565-4727. The endonuclease may comprise an HNH domainsubstantially identical to any one of SEQ ID NOs: 4565-4727.

In some cases, the endonuclease may comprise a variant having at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 55%, at least about 60%, at leastabout 65%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, or at least about 99%identity to any one of SEQ ID NOs: 930-1092. In some cases, theendonuclease may be substantially identical to any one of SEQ ID NOs:930-1092. In some cases, the endonuclease may comprise a variant havingat least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, or at leastabout 99% identity to any one of SEQ ID NOs: 930-1092. In some cases,the endonuclease may be substantially identical to any one of SEQ IDNOs: 930-1092.

In some cases, the endonuclease may comprise a variant having one ormore nuclear localization sequences (NLSs). The NLS may be proximal tothe N- or C-terminus of said endonuclease. The NLS may be appendedN-terminal or C-terminal to any one of SEQ ID NOs: 930-1092, or to avariant having at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 55%, atleast about 60%, at least about 65%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99% identity to any one of SEQ ID NOs: 930-1092.The NLS may be an SV40 large T antigen NLS. The NLS may be a c-myc NLS.The NLS can comprise a sequence with at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 99% identityto any one of SEQ ID NOs: 5593-5608. The NLS can comprise a sequencesubstantially identical to any one of SEQ ID NOs: 5593-5608. The NLS cancomprise any of the sequences in Table 1 or a combination thereof.

In some cases, sequence identity may be determined by the BLASTP,CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology searchalgorithm. The sequence identity may be determined by the BLASTPalgorithm using parameters of a wordlength (W) of 3, an expectation (E)of 10, and using a BLOSUM62 scoring matrix setting gap costs atexistence of 11, extension of 1, and using a conditional compositionalscore matrix adjustment.

In some cases, the system above may comprise (b) at least one engineeredsynthetic guide ribonucleic acid (sgRNA) capable of forming a complexwith the endonuclease bearing a 5′ targeting region complementary to adesired cleavage sequence. In some cases, the 5′ targeting region maycomprises a PAM sequence compatible with the endonuclease. In somecases, the 5′ most nucleotide of the targeting region may be G. In somecases, the 5′ targeting region may be 15-23 nucleotides in length. Theguide sequence and the tracr sequence may be supplied as separateribonucleic acids (RNAs) or a single ribonucleic acid (RNA). The guideRNA may comprise a crRNA tracrRNA binding sequence 3′ to the targetingregion. The guide RNA may comprise a tracrRNA sequence preceded by a4-nucleotide linker 3′ to the crRNA tracrRNA binding region. The sgRNAmay comprise, from 5′ to 3′: a non-natural guide nucleic acid sequencecapable of hybridizing to a target sequence in a cell; and a tracrsequence. In some cases, the non-natural guide nucleic acid sequence andthe tracr sequence are covalently linked.

In some cases, the tracr sequence may have a particular sequence. Thetracr sequence may have at least about 80% to at least about 60-100(e.g., at least about 60, at least about 65, at least about 70, at leastabout 75, at least about 80, at least about 85, or at least about 90)consecutive nucleotides of a natural tracrRNA sequence. The tracrsequence may have at least about 80% sequence identity to at least about60-100 (e.g., at least about 60, at least about 65, at least about 70,at least about 75, at least about 80, at least about 85, or at leastabout 90) consecutive nucleotides of SEQ ID NO: 5506. In some cases, thetracrRNA may have at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% identity to at leastabout 60-90 (e.g., at least about 60, at least about 65, at least about70, at least about 75, at least about 80, at least about 85, or at leastabout 90) consecutive nucleotides of SEQ ID NO: 5506. In some cases, thetracrRNA may be substantially identical to at least about 60-100 (e.g.,at least about 60, at least about 65, at least about 70, at least about75, at least about 80, at least about 85, or at least about 90)consecutive nucleotides of SEQ ID NO: 5506. The tracrRNA may compriseSEQ ID NO: 5506.

In some cases, the at least one engineered synthetic guide ribonucleicacid (sgRNA) capable of forming a complex with the endonuclease maycomprise a sequence having at least about 80% identity to SEQ ID NO:5470. The sgRNA may comprise a sequence having at least about 80%, atleast about 85%, at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, or at leastabout 99% identity to SEQ ID NO: 5470. The sgRNA may comprise a sequencesubstantially identical to SEQ ID NO: 5470.

In some cases, the system above may comprise two different sgRNAstargeting a first region and a second region for cleavage in a targetDNA locus, wherein the second region is 3′ to the first region. In somecases, the system above may comprise a single- or double-stranded DNArepair template comprising from 5′ to 3′: a first homology armcomprising a sequence of at least about 20 (e.g., at least about 40, 80,120, 150, 200, 300, 500, or 1 kb) nucleotides 5′ to the first region, asynthetic DNA sequence of at least about 10 nucleotides, and a secondhomology arm comprising a sequence of at least about 20 (e.g., at leastabout 40, 80, 120, 150, 200, 300, 500, or 1 kb) nucleotides 3′ to thesecond region.

In another aspect, the present disclosure provides a method formodifying a target nucleic acid locus of interest. The method maycomprise delivering to the target nucleic acid locus any of thenon-natural systems disclosed herein, including an enzyme and at leastone synthetic guide RNA (sgRNA) disclosed herein. The enzyme may form acomplex with the at least one sgRNA, and upon binding of the complex tothe target nucleic acid locus of interest, may modify the target nucleicacid locus of interest. Delivering the enzyme to said locus may comprisetransfecting a cell with the system or nucleic acids encoding thesystem. Delivering the nuclease to said locus may compriseelectroporating a cell with the system or nucleic acids encoding thesystem. Delivering the nuclease to said locus may comprise incubatingthe system in a buffer with a nucleic acid comprising the locus ofinterest. In some cases, the target nucleic acid locus comprisesdeoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The targetnucleic acid locus may comprise genomic DNA, viral DNA, viral RNA, orbacterial DNA. The target nucleic acid locus may be within a cell. Thetarget nucleic acid locus may be in vitro. The target nucleic acid locusmay be within a eukaryotic cell or a prokaryotic cell. The cell may bean animal cell, a human cell, bacterial cell, archaeal cell, or a plantcell. The enzyme may induce a single or double-stranded break at orproximal to the target locus of interest.

In cases where the target nucleic acid locus may be within a cell, theenzyme may be supplied as a nucleic acid containing an open readingframe encoding the enzyme having a RuvC_III domain having at least about75% (e.g., at least about 90%, at least about 91%, at least about 92%,at least about 93%, at least about 94%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, at least about 99%)identity to any one of SEQ ID NOs: 2751-2913. The deoxyribonucleic acid(DNA) containing an open reading frame encoding said endonuclease maycomprise a sequence substantially identical to SEQ ID NO: 5582 or atvariant having at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 55%, atleast about 60%, at least about 65%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99% identity to SEQ ID NO: 5582. In some cases,the nucleic acid comprises a promoter to which the open reading frameencoding the endonuclease is operably linked. The promoter may be a CMV,EF1a, SV40, PGK1, Ubc, human beta actin, CAG, TRE, or CaMKIIa promoter.The endonuclease may be supplied as a capped mRNA containing said openreading frame encoding said endonuclease. The endonuclease may besupplied as a translated polypeptide. The at least one engineered sgRNAmay be supplied as deoxyribonucleic acid (DNA) containing a genesequence encoding said at least one engineered sgRNA operably linked toa ribonucleic acid (RNA) pol III promoter. In some cases, the organismmay be eukaryotic. In some cases, the organism may be fungal. In somecases, the organism may be human.

MG16 Enzymes

In one aspect, the present disclosure provides for an engineerednuclease system comprising (a) an endonuclease. In some cases, theendonuclease is a Cas endonuclease. In some cases, the endonuclease is aType II, Class II Cas endonuclease. The endonuclease may comprise aRuvC_III domain, wherein said RuvC_III domain has at least about 70%sequence identity to any one of SEQ ID NOs: 2914-3174. In some cases,the endonuclease may comprise a RuvC_III domain, wherein the RuvC_IIIdomain has at least about 20%, at least about 25%, at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 91%, at least about 92%,at least about 93%, at least about 94%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, at least about 99%identity to any one of SEQ ID NOs: 2914-3174. In some cases, theendonuclease may comprise a RuvC_III domain, wherein the substantiallyidentical to any one of SEQ ID NOs: 2914-3174. The endonuclease maycomprise a RuvC_III domain having at least about 70% sequence identityto any one of SEQ ID NOs: 2914-3174. In some cases, the endonuclease maycomprise a RuvC_III domain having at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about91%, at least about 92%, at least about 93%, at least about 94%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, at least about 99% identity to any one of SEQ ID NOs: 2914-3174. Insome cases, the endonuclease may comprise a RuvC_III domainsubstantially identical to any one of SEQ ID NOs: 2914-3174.

The endonuclease may comprise an HNH domain having at least about 70%identity to any one of SEQ ID NOs: 4728-4988. In some cases, theendonuclease may comprise an HNH domain having at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% identical to any one ofSEQ ID NOs: 4728-4988. The endonuclease may comprise an HNH domainsubstantially identical to any one of SEQ ID NOs: 4728-4988. Theendonuclease may comprise an HNH domain having at least about 70%identity to any one of SEQ ID NOs: 4728-4988. In some cases, theendonuclease may comprise an HNH domain having at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% identical to any one ofSEQ ID NOs: 4728-4988. The endonuclease may comprise an HNH domainsubstantially identical to any one of SEQ ID NOs: 4728-4988.

In some cases, the endonuclease may comprise a variant having at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 55%, at least about 60%, at leastabout 65%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, or at least about 99%identity to any one of SEQ ID NOs: 1093-1353. In some cases, theendonuclease may be substantially identical to any one of SEQ ID NOs:1093-1353. In some cases, the endonuclease may comprise a variant havingat least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, or at leastabout 99% identity to any one of SEQ ID NOs: 1093-1353. In some cases,the endonuclease may be substantially identical to any one of SEQ IDNOs: 1093-1353.

In some cases, the endonuclease may comprise a variant having one ormore nuclear localization sequences (NLSs). The NLS may be proximal tothe N- or C-terminus of said endonuclease. The NLS may be appendedN-terminal or C-terminal to any one of SEQ ID NOs: 1093-1353, or to avariant having at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 55%, atleast about 60%, at least about 65%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99% identity to any one of SEQ ID NOs: 1093-1353.The NLS may be an SV40 large T antigen NLS. The NLS may be a c-myc NLS.The NLS can comprise a sequence with at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 99% identityto any one of SEQ ID NOs: 5593-5608. The NLS can comprise a sequencesubstantially identical to any one of SEQ ID NOs: 5593-5608. The NLS cancomprise any of the sequences in Table 1 or a combination thereof.

In some cases, sequence identity may be determined by the BLASTP,CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology searchalgorithm. The sequence identity may be determined by the BLASTPalgorithm using parameters of a wordlength (W) of 3, an expectation (E)of 10, and using a BLOSUM62 scoring matrix setting gap costs atexistence of 11, extension of 1, and using a conditional compositionalscore matrix adjustment.

In some cases, the system above may comprise (b) at least one engineeredsynthetic guide ribonucleic acid (sgRNA) capable of forming a complexwith the endonuclease bearing a 5′ targeting region complementary to adesired cleavage sequence. In some cases, the 5′ targeting region maycomprises a PAM sequence compatible with the endonuclease. In somecases, the 5′ most nucleotide of the targeting region may be G. In somecases, the 5′ targeting region may be 15-23 nucleotides in length. Theguide sequence and the tracr sequence may be supplied as separateribonucleic acids (RNAs) or a single ribonucleic acid (RNA). The guideRNA may comprise a crRNA tracrRNA binding sequence 3′ to the targetingregion. The guide RNA may comprise a tracrRNA sequence preceded by a4-nucleotide linker 3′ to the crRNA tracrRNA binding region. The sgRNAmay comprise, from 5′ to 3′: a non-natural guide nucleic acid sequencecapable of hybridizing to a target sequence in a cell; and a tracrsequence. In some cases, the non-natural guide nucleic acid sequence andthe tracr sequence are covalently linked.

In some cases, the tracr sequence may have a particular sequence. Thetracr sequence may have at least about 80% to at least about 60-100(e.g., at least about 60, at least about 65, at least about 70, at leastabout 75, at least about 80, at least about 85, or at least about 90)consecutive nucleotides of a natural tracrRNA sequence. The tracrsequence may have at least about 80% sequence identity to at least about60-100 (e.g., at least about 60, at least about 65, at least about 70,at least about 75, at least about 80, at least about 85, or at leastabout 90) consecutive nucleotides of SEQ ID NO: 5507. In some cases, thetracrRNA may have at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% identity to at leastabout 60-90 (e.g., at least about 60, at least about 65, at least about70, at least about 75, at least about 80, at least about 85, or at leastabout 90) consecutive nucleotides of SEQ ID NO: 5507. In some cases, thetracrRNA may be substantially identical to at least about 60-100 (e.g.,at least about 60, at least about 65, at least about 70, at least about75, at least about 80, at least about 85, or at least about 90)consecutive nucleotides of SEQ ID NO: 5507. The tracrRNA may compriseSEQ ID NO: 5507.

In some cases, the at least one engineered synthetic guide ribonucleicacid (sgRNA) capable of forming a complex with the endonuclease maycomprise a sequence having at least about 80% identity to SEQ ID NO:5471. The sgRNA may comprise a sequence having at least about 80%, atleast about 85%, at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, or at leastabout 99% identity to SEQ ID NO: 5471. The sgRNA may comprise a sequencesubstantially identical to SEQ ID NO: 5471.

In some cases, the system above may comprise two different sgRNAstargeting a first region and a second region for cleavage in a targetDNA locus, wherein the second region is 3′ to the first region. In somecases, the system above may comprise a single- or double-stranded DNArepair template comprising from 5′ to 3′: a first homology armcomprising a sequence of at least about 20 (e.g., at least about 40, 80,120, 150, 200, 300, 500, or 1 kb) nucleotides 5′ to the first region, asynthetic DNA sequence of at least about 10 nucleotides, and a secondhomology arm comprising a sequence of at least about 20 (e.g., at leastabout 40, 80, 120, 150, 200, 300, 500, or 1 kb) nucleotides 3′ to thesecond region.

In another aspect, the present disclosure provides a method formodifying a target nucleic acid locus of interest. The method maycomprise delivering to the target nucleic acid locus any of thenon-natural systems disclosed herein, including an enzyme and at leastone synthetic guide RNA (sgRNA) disclosed herein. The enzyme may form acomplex with the at least one sgRNA, and upon binding of the complex tothe target nucleic acid locus of interest, may modify the target nucleicacid locus of interest. Delivering the enzyme to said locus may comprisetransfecting a cell with the system or nucleic acids encoding thesystem. Delivering the nuclease to said locus may compriseelectroporating a cell with the system or nucleic acids encoding thesystem. Delivering the nuclease to said locus may comprise incubatingthe system in a buffer with a nucleic acid comprising the locus ofinterest. In some cases, the target nucleic acid locus comprisesdeoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The targetnucleic acid locus may comprise genomic DNA, viral DNA, viral RNA, orbacterial DNA. The target nucleic acid locus may be within a cell. Thetarget nucleic acid locus may be in vitro. The target nucleic acid locusmay be within a eukaryotic cell or a prokaryotic cell. The cell may bean animal cell, a human cell, bacterial cell, archaeal cell, or a plantcell. The enzyme may induce a single or double-stranded break at orproximal to the target locus of interest.

In cases where the target nucleic acid locus may be within a cell, theenzyme may be supplied as a nucleic acid containing an open readingframe encoding the enzyme having a RuvC_III domain having at least about75% (e.g., at least about 90%, at least about 91%, at least about 92%,at least about 93%, at least about 94%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, at least about 99%)identity to any one of SEQ ID NOs: 2914-3174. The deoxyribonucleic acid(DNA) containing an open reading frame encoding said endonuclease maycomprise a sequence substantially identical to SEQ ID NO: 5583 or atvariant having at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 55%, atleast about 60%, at least about 65%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99% identity to SEQ ID NO: 5583. In some cases,the nucleic acid comprises a promoter to which the open reading frameencoding the endonuclease is operably linked. The promoter may be a CMV,EF1a, SV40, PGK1, Ubc, human beta actin, CAG, TRE, or CaMKIIa promoter.The endonuclease may be supplied as a capped mRNA containing said openreading frame encoding said endonuclease. The endonuclease may besupplied as a translated polypeptide. The at least one engineered sgRNAmay be supplied as deoxyribonucleic acid (DNA) containing a genesequence encoding said at least one engineered sgRNA operably linked toa ribonucleic acid (RNA) pol III promoter. In some cases, the organismmay be eukaryotic. In some cases, the organism may be fungal. In somecases, the organism may be human.

MG18 Enzymes

In one aspect, the present disclosure provides for an engineerednuclease system comprising (a) an endonuclease. In some cases, theendonuclease is a Cas endonuclease. In some cases, the endonuclease is aType II, Class II Cas endonuclease. The endonuclease may comprise aRuvC_III domain, wherein said RuvC_III domain has at least about 70%sequence identity to any one of SEQ ID NOs: 3175-3300. In some cases,the endonuclease may comprise a RuvC_III domain, wherein the RuvC_IIIdomain has at least about 20%, at least about 25%, at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 91%, at least about 92%,at least about 93%, at least about 94%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, at least about 99%identity to any one of SEQ ID NOs: 3175-3300. In some cases, theendonuclease may comprise a RuvC_III domain, wherein the substantiallyidentical to any one of SEQ ID NOs: 3175-3300. The endonuclease maycomprise a RuvC_III domain having at least about 70% sequence identityto any one of SEQ ID NOs: 3175-3300. In some cases, the endonuclease maycomprise a RuvC_III domain having at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about91%, at least about 92%, at least about 93%, at least about 94%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, at least about 99% identity to any one of SEQ ID NOs: 3175-3300. Insome cases, the endonuclease may comprise a RuvC_III domainsubstantially identical to any one of SEQ ID NOs: 3175-3300.

The endonuclease may comprise an HNH domain having at least about 70%identity to any one of SEQ ID NOs: 4989-5146. In some cases, theendonuclease may comprise an HNH domain having at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% identical to any one ofSEQ ID NOs: 4989-5146. The endonuclease may comprise an HNH domainsubstantially identical to any one of SEQ ID NOs: 4989-5146. Theendonuclease may comprise an HNH domain having at least about 70%identity to any one of SEQ ID NOs: 4989-5146. In some cases, theendonuclease may comprise an HNH domain having at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% identical to any one ofSEQ ID NOs: 4989-5146. The endonuclease may comprise an HNH domainsubstantially identical to any one of SEQ ID NOs: 4989-5146.

In some cases, the endonuclease may comprise a variant having at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 55%, at least about 60%, at leastabout 65%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, or at least about 99%identity to any one of SEQ ID NOs: 1354-1511. In some cases, theendonuclease may be substantially identical to any one of SEQ ID NOs:1354-1511. In some cases, the endonuclease may comprise a variant havingat least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, or at leastabout 99% identity to any one of SEQ ID NOs: 1354-1511. In some cases,the endonuclease may be substantially identical to any one of SEQ IDNOs: 1354-1511.

In some cases, the endonuclease may comprise a variant having one ormore nuclear localization sequences (NLSs). The NLS may be proximal tothe N- or C-terminus of said endonuclease. The NLS may be appendedN-terminal or C-terminal to any one of SEQ ID NOs: 1354-1511, or to avariant having at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 55%, atleast about 60%, at least about 65%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99% identity to any one of SEQ ID NOs: 1354-1511.The NLS may be an SV40 large T antigen NLS. The NLS may be a c-myc NLS.The NLS can comprise a sequence with at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 99% identityto any one of SEQ ID NOs: 5593-5608. The NLS can comprise a sequencesubstantially identical to any one of SEQ ID NOs: 5593-5608. The NLS cancomprise any of the sequences in Table 1 or a combination thereof.

In some cases, sequence identity may be determined by the BLASTP,CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology searchalgorithm. The sequence identity may be determined by the BLASTPalgorithm using parameters of a wordlength (W) of 3, an expectation (E)of 10, and using a BLOSUM62 scoring matrix setting gap costs atexistence of 11, extension of 1, and using a conditional compositionalscore matrix adjustment.

In some cases, the system above may comprise (b) at least one engineeredsynthetic guide ribonucleic acid (sgRNA) capable of forming a complexwith the endonuclease bearing a 5′ targeting region complementary to adesired cleavage sequence. In some cases, the 5′ targeting region maycomprises a PAM sequence compatible with the endonuclease. In somecases, the 5′ most nucleotide of the targeting region may be G. In somecases, the 5′ targeting region may be 15-23 nucleotides in length. Theguide sequence and the tracr sequence may be supplied as separateribonucleic acids (RNAs) or a single ribonucleic acid (RNA). The guideRNA may comprise a crRNA tracrRNA binding sequence 3′ to the targetingregion. The guide RNA may comprise a tracrRNA sequence preceded by a4-nucleotide linker 3′ to the crRNA tracrRNA binding region. The sgRNAmay comprise, from 5′ to 3′: a non-natural guide nucleic acid sequencecapable of hybridizing to a target sequence in a cell; and a tracrsequence. In some cases, the non-natural guide nucleic acid sequence andthe tracr sequence are covalently linked.

In some cases, the tracr sequence may have a particular sequence. Thetracr sequence may have at least about 80% to at least about 60-100(e.g., at least about 60, at least about 65, at least about 70, at leastabout 75, at least about 80, at least about 85, or at least about 90)consecutive nucleotides of a natural tracrRNA sequence. The tracrsequence may have at least about 80% sequence identity to at least about60-100 (e.g., at least about 60, at least about 65, at least about 70,at least about 75, at least about 80, at least about 85, or at leastabout 90) consecutive nucleotides of SEQ ID NO: 5508. In some cases, thetracrRNA may have at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% identity to at leastabout 60-90 (e.g., at least about 60, at least about 65, at least about70, at least about 75, at least about 80, at least about 85, or at leastabout 90) consecutive nucleotides of SEQ ID NO: 5508. In some cases, thetracrRNA may be substantially identical to at least about 60-100 (e.g.,at least about 60, at least about 65, at least about 70, at least about75, at least about 80, at least about 85, or at least about 90)consecutive nucleotides of SEQ ID NO: 5508. The tracrRNA may compriseSEQ ID NO: 5508.

In some cases, the at least one engineered synthetic guide ribonucleicacid (sgRNA) capable of forming a complex with the endonuclease maycomprise a sequence having at least about 80% identity to SEQ ID NO:5472. The sgRNA may comprise a sequence having at least about 80%, atleast about 85%, at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, or at leastabout 99% identity to SEQ ID NO: 5472. The sgRNA may comprise a sequencesubstantially identical to SEQ ID NO: 5472.

In some cases, the system above may comprise two different sgRNAstargeting a first region and a second region for cleavage in a targetDNA locus, wherein the second region is 3′ to the first region. In somecases, the system above may comprise a single- or double-stranded DNArepair template comprising from 5′ to 3′: a first homology armcomprising a sequence of at least about 20 (e.g., at least about 40, 80,120, 150, 200, 300, 500, or 1 kb) nucleotides 5′ to the first region, asynthetic DNA sequence of at least about 10 nucleotides, and a secondhomology arm comprising a sequence of at least about 20 (e.g., at leastabout 40, 80, 120, 150, 200, 300, 500, or 1 kb) nucleotides 3′ to thesecond region.

In another aspect, the present disclosure provides a method formodifying a target nucleic acid locus of interest. The method maycomprise delivering to the target nucleic acid locus any of thenon-natural systems disclosed herein, including an enzyme and at leastone synthetic guide RNA (sgRNA) disclosed herein. The enzyme may form acomplex with the at least one sgRNA, and upon binding of the complex tothe target nucleic acid locus of interest, may modify the target nucleicacid locus of interest. Delivering the enzyme to said locus may comprisetransfecting a cell with the system or nucleic acids encoding thesystem. Delivering the nuclease to said locus may compriseelectroporating a cell with the system or nucleic acids encoding thesystem. Delivering the nuclease to said locus may comprise incubatingthe system in a buffer with a nucleic acid comprising the locus ofinterest. In some cases, the target nucleic acid locus comprisesdeoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The targetnucleic acid locus may comprise genomic DNA, viral DNA, viral RNA, orbacterial DNA. The target nucleic acid locus may be within a cell. Thetarget nucleic acid locus may be in vitro. The target nucleic acid locusmay be within a eukaryotic cell or a prokaryotic cell. The cell may bean animal cell, a human cell, bacterial cell, archaeal cell, or a plantcell. The enzyme may induce a single or double-stranded break at orproximal to the target locus of interest.

In cases where the target nucleic acid locus may be within a cell, theenzyme may be supplied as a nucleic acid containing an open readingframe encoding the enzyme having a RuvC_III domain having at least about75% (e.g., at least about 90%, at least about 91%, at least about 92%,at least about 93%, at least about 94%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, at least about 99%)identity to any one of SEQ ID NOs: 3175-3300. The deoxyribonucleic acid(DNA) containing an open reading frame encoding said endonuclease maycomprise a sequence substantially identical to SEQ ID NOs: 5584 or atvariant having at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 55%, atleast about 60%, at least about 65%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99% identity to SEQ ID NOs: 5584. In some cases,the nucleic acid comprises a promoter to which the open reading frameencoding the endonuclease is operably linked. The promoter may be a CMV,EF1a, SV40, PGK1, Ubc, human beta actin, CAG, TRE, or CaMKIIa promoter.The endonuclease may be supplied as a capped mRNA containing said openreading frame encoding said endonuclease. The endonuclease may besupplied as a translated polypeptide. The at least one engineered sgRNAmay be supplied as deoxyribonucleic acid (DNA) containing a genesequence encoding said at least one engineered sgRNA operably linked toa ribonucleic acid (RNA) pol III promoter. In some cases, the organismmay be eukaryotic. In some cases, the organism may be fungal. In somecases, the organism may be human.

MG21 Enzymes

In one aspect, the present disclosure provides for an engineerednuclease system comprising (a) an endonuclease. In some cases, theendonuclease is a Cas endonuclease. In some cases, the endonuclease is aType II, Class II Cas endonuclease. The endonuclease may comprise aRuvC_III domain, wherein said RuvC_III domain has at least about 70%sequence identity to any one of SEQ ID NOs: 3331-3474. In some cases,the endonuclease may comprise a RuvC_III domain, wherein the RuvC_IIIdomain has at least about 20%, at least about 25%, at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 91%, at least about 92%,at least about 93%, at least about 94%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, at least about 99%identity to any one of SEQ ID NOs: 3331-3474. In some cases, theendonuclease may comprise a RuvC_III domain, wherein the substantiallyidentical to any one of SEQ ID NOs: 3331-3474. The endonuclease maycomprise a RuvC_III domain having at least about 70% sequence identityto any one of SEQ ID NOs: 3331-3474. In some cases, the endonuclease maycomprise a RuvC_III domain having at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about91%, at least about 92%, at least about 93%, at least about 94%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, at least about 99% identity to any one of SEQ ID NOs: 3331-3474. Insome cases, the endonuclease may comprise a RuvC_III domainsubstantially identical to any one of SEQ ID NOs: 3331-3474.

The endonuclease may comprise an HNH domain having at least about 70%identity to any one of SEQ ID NOs: 5147-5290. In some cases, theendonuclease may comprise an HNH domain having at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% identical to any one ofSEQ ID NOs: 5147-5290. The endonuclease may comprise an HNH domainsubstantially identical to any one of SEQ ID NOs: 5147-5290. Theendonuclease may comprise an HNH domain having at least about 70%identity to any one of SEQ ID NOs: 5147-5290. In some cases, theendonuclease may comprise an HNH domain having at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% identical to any one ofSEQ ID NOs: 5147-5290. The endonuclease may comprise an HNH domainsubstantially identical to any one of SEQ ID NOs: 5147-5290.

In some cases, the endonuclease may comprise a variant having at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 55%, at least about 60%, at leastabout 65%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, or at least about 99%identity to any one of SEQ ID NOs: 1512-1655. In some cases, theendonuclease may be substantially identical to any one of SEQ ID NOs:1512-1655. In some cases, the endonuclease may comprise a variant havingat least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, or at leastabout 99% identity to any one of SEQ ID NOs: 1512-1655. In some cases,the endonuclease may be substantially identical to any one of SEQ IDNOs: 1512-1655.

In some cases, the endonuclease may comprise a variant having one ormore nuclear localization sequences (NLSs). The NLS may be proximal tothe N- or C-terminus of said endonuclease. The NLS may be appendedN-terminal or C-terminal to any one of SEQ ID NOs: 1512-1655, or to avariant having at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 55%, atleast about 60%, at least about 65%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99% identity to any one of SEQ ID NOs: 1512-1655.The NLS may be an SV40 large T antigen NLS. The NLS may be a c-myc NLS.The NLS can comprise a sequence with at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 99% identityto any one of SEQ ID NOs: 5593-5608. The NLS can comprise a sequencesubstantially identical to any one of SEQ ID NOs: 5593-5608. The NLS cancomprise any of the sequences in Table 1 or a combination thereof.

In some cases, sequence identity may be determined by the BLASTP,CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology searchalgorithm. The sequence identity may be determined by the BLASTPalgorithm using parameters of a wordlength (W) of 3, an expectation (E)of 10, and using a BLOSUM62 scoring matrix setting gap costs atexistence of 11, extension of 1, and using a conditional compositionalscore matrix adjustment.

In some cases, the system above may comprise (b) at least one engineeredsynthetic guide ribonucleic acid (sgRNA) capable of forming a complexwith the endonuclease bearing a 5′ targeting region complementary to adesired cleavage sequence. In some cases, the 5′ targeting region maycomprises a PAM sequence compatible with the endonuclease. In somecases, the 5′ most nucleotide of the targeting region may be G. In somecases, the 5′ targeting region may be 15-23 nucleotides in length. Theguide sequence and the tracr sequence may be supplied as separateribonucleic acids (RNAs) or a single ribonucleic acid (RNA). The guideRNA may comprise a crRNA tracrRNA binding sequence 3′ to the targetingregion. The guide RNA may comprise a tracrRNA sequence preceded by a4-nucleotide linker 3′ to the crRNA tracrRNA binding region. The sgRNAmay comprise, from 5′ to 3′: a non-natural guide nucleic acid sequencecapable of hybridizing to a target sequence in a cell; and a tracrsequence. In some cases, the non-natural guide nucleic acid sequence andthe tracr sequence are covalently linked.

In some cases, the tracr sequence may have a particular sequence. Thetracr sequence may have at least about 80% to at least about 60-100(e.g., at least about 60, at least about 65, at least about 70, at leastabout 75, at least about 80, at least about 85, or at least about 90)consecutive nucleotides of a natural tracrRNA sequence. The tracrsequence may have at least about 80% sequence identity to at least about60-100 (e.g., at least about 60, at least about 65, at least about 70,at least about 75, at least about 80, at least about 85, or at leastabout 90) consecutive nucleotides of SEQ ID NO: 5509. In some cases, thetracrRNA may have at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% identity to at leastabout 60-90 (e.g., at least about 60, at least about 65, at least about70, at least about 75, at least about 80, at least about 85, or at leastabout 90) consecutive nucleotides of SEQ ID NO: 5509. In some cases, thetracrRNA may be substantially identical to at least about 60-100 (e.g.,at least about 60, at least about 65, at least about 70, at least about75, at least about 80, at least about 85, or at least about 90)consecutive nucleotides of SEQ ID NO: 5509. The tracrRNA may compriseSEQ ID NO: 5509.

In some cases, the at least one engineered synthetic guide ribonucleicacid (sgRNA) capable of forming a complex with the endonuclease maycomprise a sequence having at least about 80% identity to SEQ ID NO:5473. The sgRNA may comprise a sequence having at least about 80%, atleast about 85%, at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, or at leastabout 99% identity to SEQ ID NO: 5473. The sgRNA may comprise a sequencesubstantially identical to SEQ ID NO: 5473.

In some cases, the system above may comprise two different sgRNAstargeting a first region and a second region for cleavage in a targetDNA locus, wherein the second region is 3′ to the first region. In somecases, the system above may comprise a single- or double-stranded DNArepair template comprising from 5′ to 3′: a first homology armcomprising a sequence of at least about 20 (e.g., at least about 40, 80,120, 150, 200, 300, 500, or 1 kb) nucleotides 5′ to the first region, asynthetic DNA sequence of at least about 10 nucleotides, and a secondhomology arm comprising a sequence of at least about 20 (e.g., at leastabout 40, 80, 120, 150, 200, 300, 500, or 1 kb) nucleotides 3′ to thesecond region.

In another aspect, the present disclosure provides a method formodifying a target nucleic acid locus of interest. The method maycomprise delivering to the target nucleic acid locus any of thenon-natural systems disclosed herein, including an enzyme and at leastone synthetic guide RNA (sgRNA) disclosed herein. The enzyme may form acomplex with the at least one sgRNA, and upon binding of the complex tothe target nucleic acid locus of interest, may modify the target nucleicacid locus of interest. Delivering the enzyme to said locus may comprisetransfecting a cell with the system or nucleic acids encoding thesystem. Delivering the nuclease to said locus may compriseelectroporating a cell with the system or nucleic acids encoding thesystem. Delivering the nuclease to said locus may comprise incubatingthe system in a buffer with a nucleic acid comprising the locus ofinterest. In some cases, the target nucleic acid locus comprisesdeoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The targetnucleic acid locus may comprise genomic DNA, viral DNA, viral RNA, orbacterial DNA. The target nucleic acid locus may be within a cell. Thetarget nucleic acid locus may be in vitro. The target nucleic acid locusmay be within a eukaryotic cell or a prokaryotic cell. The cell may bean animal cell, a human cell, bacterial cell, archaeal cell, or a plantcell. The enzyme may induce a single or double-stranded break at orproximal to the target locus of interest.

In cases where the target nucleic acid locus may be within a cell, theenzyme may be supplied as a nucleic acid containing an open readingframe encoding the enzyme having a RuvC_III domain having at least about75% (e.g., at least about 90%, at least about 91%, at least about 92%,at least about 93%, at least about 94%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, at least about 99%)identity to any one of SEQ ID NOs: 3331-3474. The deoxyribonucleic acid(DNA) containing an open reading frame encoding said endonuclease maycomprise a sequence substantially identical to SEQ ID NOs: 5585 or atvariant having at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 55%, atleast about 60%, at least about 65%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99% identity to SEQ ID NOs: 5585. In some cases,the nucleic acid comprises a promoter to which the open reading frameencoding the endonuclease is operably linked. The promoter may be a CMV,EF1a, SV40, PGK1, Ubc, human beta actin, CAG, TRE, or CaMKIIa promoter.The endonuclease may be supplied as a capped mRNA containing said openreading frame encoding said endonuclease. The endonuclease may besupplied as a translated polypeptide. The at least one engineered sgRNAmay be supplied as deoxyribonucleic acid (DNA) containing a genesequence encoding said at least one engineered sgRNA operably linked toa ribonucleic acid (RNA) pol III promoter. In some cases, the organismmay be eukaryotic. In some cases, the organism may be fungal. In somecases, the organism may be human.

MG22 Enzymes

In one aspect, the present disclosure provides for an engineerednuclease system comprising (a) an endonuclease. In some cases, theendonuclease is a Cas endonuclease. In some cases, the endonuclease is aType II, Class II Cas endonuclease. The endonuclease may comprise aRuvC_III domain, wherein said RuvC_III domain has at least about 70%sequence identity to any one of SEQ ID NOs: 3475-3568. In some cases,the endonuclease may comprise a RuvC_III domain, wherein the RuvC_IIIdomain has at least about 20%, at least about 25%, at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 91%, at least about 92%,at least about 93%, at least about 94%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, at least about 99%identity to any one of SEQ ID NOs: 3475-3568. In some cases, theendonuclease may comprise a RuvC_III domain, wherein the substantiallyidentical to any one of SEQ ID NOs: 3475-3568. The endonuclease maycomprise a RuvC_III domain having at least about 70% sequence identityto any one of SEQ ID NOs: 3475-3568. In some cases, the endonuclease maycomprise a RuvC_III domain having at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about91%, at least about 92%, at least about 93%, at least about 94%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, at least about 99% identity to any one of SEQ ID NOs: 3475-3568. Insome cases, the endonuclease may comprise a RuvC_III domainsubstantially identical to any one of SEQ ID NOs: 3475-3568.

The endonuclease may comprise an HNH domain having at least about 70%identity to any one of SEQ ID NOs: 5291-5389. In some cases, theendonuclease may comprise an HNH domain having at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% identical to any one ofSEQ ID NOs: 5291-5389. The endonuclease may comprise an HNH domainsubstantially identical to any one of SEQ ID NOs: 5291-5389. Theendonuclease may comprise an HNH domain having at least about 70%identity to any one of SEQ ID NOs: 5291-5389. In some cases, theendonuclease may comprise an HNH domain having at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% identical to any one ofSEQ ID NOs: 5291-5389. The endonuclease may comprise an HNH domainsubstantially identical to any one of SEQ ID NOs: 5291-5389.

In some cases, the endonuclease may comprise a variant having at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 55%, at least about 60%, at leastabout 65%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, or at least about 99%identity to any one of SEQ ID NOs: 1656-1755. In some cases, theendonuclease may be substantially identical to any one of SEQ ID NOs:1656-1755. In some cases, the endonuclease may comprise a variant havingat least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, or at leastabout 99% identity to any one of SEQ ID NOs: 1656-1755. In some cases,the endonuclease may be substantially identical to any one of SEQ IDNOs: 1656-1755.

In some cases, the endonuclease may comprise a variant having one ormore nuclear localization sequences (NLSs). The NLS may be proximal tothe N- or C-terminus of said endonuclease. The NLS may be appendedN-terminal or C-terminal to any one of SEQ ID NOs: 432-660, or to avariant having at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 55%, atleast about 60%, at least about 65%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99% identity to any one of SEQ ID NOs: 1656-1755.The NLS may be an SV40 large T antigen NLS. The NLS may be a c-myc NLS.The NLS can comprise a sequence with at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 99% identityto any one of SEQ ID NOs: 5593-5608. The NLS can comprise a sequencesubstantially identical to any one of SEQ ID NOs: 5593-5608. The NLS cancomprise any of the sequences in Table 1 or a combination thereof.

In some cases, sequence identity may be determined by the BLASTP,CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology searchalgorithm. The sequence identity may be determined by the BLASTPalgorithm using parameters of a wordlength (W) of 3, an expectation (E)of 10, and using a BLOSUM62 scoring matrix setting gap costs atexistence of 11, extension of 1, and using a conditional compositionalscore matrix adjustment.

In some cases, the system above may comprise (b) at least one engineeredsynthetic guide ribonucleic acid (sgRNA) capable of forming a complexwith the endonuclease bearing a 5′ targeting region complementary to adesired cleavage sequence. In some cases, the 5′ targeting region maycomprises a PAM sequence compatible with the endonuclease. In somecases, the 5′ most nucleotide of the targeting region may be G. In somecases, the 5′ targeting region may be 15-23 nucleotides in length. Theguide sequence and the tracr sequence may be supplied as separateribonucleic acids (RNAs) or a single ribonucleic acid (RNA). The guideRNA may comprise a crRNA tracrRNA binding sequence 3′ to the targetingregion. The guide RNA may comprise a tracrRNA sequence preceded by a4-nucleotide linker 3′ to the crRNA tracrRNA binding region. The sgRNAmay comprise, from 5′ to 3′: a non-natural guide nucleic acid sequencecapable of hybridizing to a target sequence in a cell; and a tracrsequence. In some cases, the non-natural guide nucleic acid sequence andthe tracr sequence are covalently linked.

In some cases, the tracr sequence may have a particular sequence. Thetracr sequence may have at least about 80% to at least about 60-100(e.g., at least about 60, at least about 65, at least about 70, at leastabout 75, at least about 80, at least about 85, or at least about 90)consecutive nucleotides of a natural tracrRNA sequence. The tracrsequence may have at least about 80% sequence identity to at least about60-100 (e.g., at least about 60, at least about 65, at least about 70,at least about 75, at least about 80, at least about 85, or at leastabout 90) consecutive nucleotides of SEQ ID NO: 5510. In some cases, thetracrRNA may have at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% identity to at leastabout 60-90 (e.g., at least about 60, at least about 65, at least about70, at least about 75, at least about 80, at least about 85, or at leastabout 90) consecutive nucleotides of SEQ ID NO: 5510. In some cases, thetracrRNA may be substantially identical to at least about 60-100 (e.g.,at least about 60, at least about 65, at least about 70, at least about75, at least about 80, at least about 85, or at least about 90)consecutive nucleotides of SEQ ID NO: 5510. The tracrRNA may compriseSEQ ID NO: 5510.

In some cases, the at least one engineered synthetic guide ribonucleicacid (sgRNA) capable of forming a complex with the endonuclease maycomprise a sequence having at least about 80% identity to SEQ ID NO:5474. The sgRNA may comprise a sequence having at least about 80%, atleast about 85%, at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, or at leastabout 99% identity to SEQ ID NO: 5474. The sgRNA may comprise a sequencesubstantially identical to SEQ ID NO: 5474.

In some cases, the system above may comprise two different sgRNAstargeting a first region and a second region for cleavage in a targetDNA locus, wherein the second region is 3′ to the first region. In somecases, the system above may comprise a single- or double-stranded DNArepair template comprising from 5′ to 3′: a first homology armcomprising a sequence of at least about 20 (e.g., at least about 40, 80,120, 150, 200, 300, 500, or 1 kb) nucleotides 5′ to the first region, asynthetic DNA sequence of at least about 10 nucleotides, and a secondhomology arm comprising a sequence of at least about 20 (e.g., at leastabout 40, 80, 120, 150, 200, 300, 500, or 1 kb) nucleotides 3′ to thesecond region.

In another aspect, the present disclosure provides a method formodifying a target nucleic acid locus of interest. The method maycomprise delivering to the target nucleic acid locus any of thenon-natural systems disclosed herein, including an enzyme and at leastone synthetic guide RNA (sgRNA) disclosed herein. The enzyme may form acomplex with the at least one sgRNA, and upon binding of the complex tothe target nucleic acid locus of interest, may modify the target nucleicacid locus of interest. Delivering the enzyme to said locus may comprisetransfecting a cell with the system or nucleic acids encoding thesystem. Delivering the nuclease to said locus may compriseelectroporating a cell with the system or nucleic acids encoding thesystem. Delivering the nuclease to said locus may comprise incubatingthe system in a buffer with a nucleic acid comprising the locus ofinterest. In some cases, the target nucleic acid locus comprisesdeoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The targetnucleic acid locus may comprise genomic DNA, viral DNA, viral RNA, orbacterial DNA. The target nucleic acid locus may be within a cell. Thetarget nucleic acid locus may be in vitro. The target nucleic acid locusmay be within a eukaryotic cell or a prokaryotic cell. The cell may bean animal cell, a human cell, bacterial cell, archaeal cell, or a plantcell. The enzyme may induce a single or double-stranded break at orproximal to the target locus of interest.

In cases where the target nucleic acid locus may be within a cell, theenzyme may be supplied as a nucleic acid containing an open readingframe encoding the enzyme having a RuvC_III domain having at least about75% (e.g., at least about 90%, at least about 91%, at least about 92%,at least about 93%, at least about 94%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, at least about 99%)identity to any one of SEQ ID NOs: 3475-3568. The deoxyribonucleic acid(DNA) containing an open reading frame encoding said endonuclease maycomprise a sequence substantially identical to SEQ ID NOs: 5586 or atvariant having at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 55%, atleast about 60%, at least about 65%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99% identity to SEQ ID NOs: 5586. In some cases,the nucleic acid comprises a promoter to which the open reading frameencoding the endonuclease is operably linked. The promoter may be a CMV,EF1a, SV40, PGK1, Ubc, human beta actin, CAG, TRE, or CaMKIIa promoter.The endonuclease may be supplied as a capped mRNA containing said openreading frame encoding said endonuclease. The endonuclease may besupplied as a translated polypeptide. The at least one engineered sgRNAmay be supplied as deoxyribonucleic acid (DNA) containing a genesequence encoding said at least one engineered sgRNA operably linked toa ribonucleic acid (RNA) pol III promoter. In some cases, the organismmay be eukaryotic. In some cases, the organism may be fungal. In somecases, the organism may be human.

MG23 Enzymes

In one aspect, the present disclosure provides for an engineerednuclease system comprising (a) an endonuclease. In some cases, theendonuclease is a Cas endonuclease. In some cases, the endonuclease is aType II, Class II Cas endonuclease. The endonuclease may comprise aRuvC_III domain, wherein said RuvC_III domain has at least about 70%sequence identity to any one of SEQ ID NOs: 3569-3637. In some cases,the endonuclease may comprise a RuvC_III domain, wherein the RuvC_IIIdomain has at least about 20%, at least about 25%, at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 91%, at least about 92%,at least about 93%, at least about 94%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, at least about 99%identity to any one of SEQ ID NOs: 3569-3637. In some cases, theendonuclease may comprise a RuvC_III domain, wherein the substantiallyidentical to any one of SEQ ID NOs: 3569-3637. The endonuclease maycomprise a RuvC_III domain having at least about 70% sequence identityto any one of SEQ ID NOs: 3569-3637. In some cases, the endonuclease maycomprise a RuvC_III domain having at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about91%, at least about 92%, at least about 93%, at least about 94%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, at least about 99% identity to any one of SEQ ID NOs: 3569-3637. Insome cases, the endonuclease may comprise a RuvC_III domainsubstantially identical to any one of SEQ ID NOs: 3569-3637.

The endonuclease may comprise an HNH domain having at least about 70%identity to any one of SEQ ID NOs: 5390-5460. In some cases, theendonuclease may comprise an HNH domain having at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% identical to any one ofSEQ ID NOs: 5390-5460. The endonuclease may comprise an HNH domainsubstantially identical to any one of SEQ ID NOs: 5390-5460. Theendonuclease may comprise an HNH domain having at least about 70%identity to any one of SEQ ID NOs: 5390-5460. In some cases, theendonuclease may comprise an HNH domain having at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% identical to any one ofSEQ ID NOs: 5390-5460. The endonuclease may comprise an HNH domainsubstantially identical to any one of SEQ ID NOs: 5390-5460.

In some cases, the endonuclease may comprise a variant having at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 55%, at least about 60%, at leastabout 65%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, or at least about 99%identity to any one of SEQ ID NOs: 1756-1826. In some cases, theendonuclease may be substantially identical to any one of SEQ ID NOs:1756-1826. In some cases, the endonuclease may comprise a variant havingat least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, or at leastabout 99% identity to any one of SEQ ID NOs: 1756-1826. In some cases,the endonuclease may be substantially identical to any one of SEQ IDNOs: 1756-1826.

In some cases, the endonuclease may comprise a variant having one ormore nuclear localization sequences (NLSs). The NLS may be proximal tothe N- or C-terminus of said endonuclease. The NLS may be appendedN-terminal or C-terminal to any one of SEQ ID NOs: 1756-1826, or to avariant having at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 55%, atleast about 60%, at least about 65%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99% identity to any one of SEQ ID NOs: 1756-1826.The NLS may be an SV40 large T antigen NLS. The NLS may be a c-myc NLS.The NLS can comprise a sequence with at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 99% identityto any one of SEQ ID NOs: 5593-5608. The NLS can comprise a sequencesubstantially identical to any one of SEQ ID NOs: 5593-5608. The NLS cancomprise any of the sequences in Table 1 or a combination thereof.

In some cases, sequence identity may be determined by the BLASTP,CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology searchalgorithm. The sequence identity may be determined by the BLASTPalgorithm using parameters of a wordlength (W) of 3, an expectation (E)of 10, and using a BLOSUM62 scoring matrix setting gap costs atexistence of 11, extension of 1, and using a conditional compositionalscore matrix adjustment.

In some cases, the system above may comprise (b) at least one engineeredsynthetic guide ribonucleic acid (sgRNA) capable of forming a complexwith the endonuclease bearing a 5′ targeting region complementary to adesired cleavage sequence. In some cases, the 5′ targeting region maycomprises a PAM sequence compatible with the endonuclease. In somecases, the 5′ most nucleotide of the targeting region may be G. In somecases, the 5′ targeting region may be 15-23 nucleotides in length. Theguide sequence and the tracr sequence may be supplied as separateribonucleic acids (RNAs) or a single ribonucleic acid (RNA). The guideRNA may comprise a crRNA tracrRNA binding sequence 3′ to the targetingregion. The guide RNA may comprise a tracrRNA sequence preceded by a4-nucleotide linker 3′ to the crRNA tracrRNA binding region. The sgRNAmay comprise, from 5′ to 3′: a non-natural guide nucleic acid sequencecapable of hybridizing to a target sequence in a cell; and a tracrsequence. In some cases, the non-natural guide nucleic acid sequence andthe tracr sequence are covalently linked.

In some cases, the tracr sequence may have a particular sequence. Thetracr sequence may have at least about 80% to at least about 60-100(e.g., at least about 60, at least about 65, at least about 70, at leastabout 75, at least about 80, at least about 85, or at least about 90)consecutive nucleotides of a natural tracrRNA sequence. The tracrsequence may have at least about 80% sequence identity to at least about60-100 (e.g., at least about 60, at least about 65, at least about 70,at least about 75, at least about 80, at least about 85, or at leastabout 90) consecutive nucleotides of SEQ ID NO: 5511. In some cases, thetracrRNA may have at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% identity to at leastabout 60-90 (e.g., at least about 60, at least about 65, at least about70, at least about 75, at least about 80, at least about 85, or at leastabout 90) consecutive nucleotides of SEQ ID NO: 5511. In some cases, thetracrRNA may be substantially identical to at least about 60-100 (e.g.,at least about 60, at least about 65, at least about 70, at least about75, at least about 80, at least about 85, or at least about 90)consecutive nucleotides of SEQ ID NO: 5511. The tracrRNA may compriseSEQ ID NO: 5511.

In some cases, the at least one engineered synthetic guide ribonucleicacid (sgRNA) capable of forming a complex with the endonuclease maycomprise a sequence having at least about 80% identity to SEQ ID NO:5475. The sgRNA may comprise a sequence having at least about 80%, atleast about 85%, at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, or at leastabout 99% identity to SEQ ID NO: 5475. The sgRNA may comprise a sequencesubstantially identical to SEQ ID NO: 5475.

In some cases, the system above may comprise two different sgRNAstargeting a first region and a second region for cleavage in a targetDNA locus, wherein the second region is 3′ to the first region. In somecases, the system above may comprise a single- or double-stranded DNArepair template comprising from 5′ to 3′: a first homology armcomprising a sequence of at least about 20 (e.g., at least about 40, 80,120, 150, 200, 300, 500, or 1 kb) nucleotides 5′ to the first region, asynthetic DNA sequence of at least about 10 nucleotides, and a secondhomology arm comprising a sequence of at least about 20 (e.g., at leastabout 40, 80, 120, 150, 200, 300, 500, or 1 kb) nucleotides 3′ to thesecond region.

In another aspect, the present disclosure provides a method formodifying a target nucleic acid locus of interest. The method maycomprise delivering to the target nucleic acid locus any of thenon-natural systems disclosed herein, including an enzyme and at leastone synthetic guide RNA (sgRNA) disclosed herein. The enzyme may form acomplex with the at least one sgRNA, and upon binding of the complex tothe target nucleic acid locus of interest, may modify the target nucleicacid locus of interest. Delivering the enzyme to said locus may comprisetransfecting a cell with the system or nucleic acids encoding thesystem. Delivering the nuclease to said locus may compriseelectroporating a cell with the system or nucleic acids encoding thesystem. Delivering the nuclease to said locus may comprise incubatingthe system in a buffer with a nucleic acid comprising the locus ofinterest. In some cases, the target nucleic acid locus comprisesdeoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The targetnucleic acid locus may comprise genomic DNA, viral DNA, viral RNA, orbacterial DNA. The target nucleic acid locus may be within a cell. Thetarget nucleic acid locus may be in vitro. The target nucleic acid locusmay be within a eukaryotic cell or a prokaryotic cell. The cell may bean animal cell, a human cell, bacterial cell, archaeal cell, or a plantcell. The enzyme may induce a single or double-stranded break at orproximal to the target locus of interest.

In cases where the target nucleic acid locus may be within a cell, theenzyme may be supplied as a nucleic acid containing an open readingframe encoding the enzyme having a RuvC_III domain having at least about75% (e.g., at least about 90%, at least about 91%, at least about 92%,at least about 93%, at least about 94%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, at least about 99%)identity to any one of SEQ ID NOs: 3569-3637. The deoxyribonucleic acid(DNA) containing an open reading frame encoding said endonuclease maycomprise a sequence substantially identical to SEQ ID NOs: 5587 or atvariant having at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 55%, atleast about 60%, at least about 65%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99% identity to SEQ ID NOs: 5587. In some cases,the nucleic acid comprises a promoter to which the open reading frameencoding the endonuclease is operably linked. The promoter may be a CMV,EF1a, SV40, PGK1, Ubc, human beta actin, CAG, TRE, or CaMKIIa promoter.The endonuclease may be supplied as a capped mRNA containing said openreading frame encoding said endonuclease. The endonuclease may besupplied as a translated polypeptide. The at least one engineered sgRNAmay be supplied as deoxyribonucleic acid (DNA) containing a genesequence encoding said at least one engineered sgRNA operably linked toa ribonucleic acid (RNA) pol III promoter. In some cases, the organismmay be eukaryotic. In some cases, the organism may be fungal. In somecases, the organism may be human.

EXAMPLES Example 1.—Metagenomic Analysis for New Proteins

Metagenomic samples were collected from sediment, soil and animal.Deoxyribonucleic acid (DNA) was extracted with a Zymobiomics DNAmini-prep kit and sequenced on an Illumina HiSeq® 2500. Samples werecollected with consent of property owners. Additional raw sequence datafrom public sources included animal microbiomes, sediment, soil, hotsprings, hydrothermal vents, marine, peat bogs, permafrost, and sewagesequences. Metagenomic sequence data was searched using Hidden MarkovModels generated based on known Cas protein sequences including type IICas effector proteins. Novel effector proteins identified by the searchwere aligned to known proteins to identify potential active sites. Thismetagenomic workflow resulted in delineation of the MG1, MG2, MG3, MG4,MG6, MG14, MG15, MG16, MG18, MG21, MG22, and MG23 families of class II,type II CRISPR endonucleases described herein.

Example 2A.—Discovery of an MG1 Family of CRISPR Systems

Analysis of the data from the metagenomic analysis of Example 1 revealeda new cluster of previously undescribed putative CRISPR systemsinitially comprising six members (MG1-1, MG1-2, MG1-3, MG1-4, MG1-5, andMG1-6 recorded as SEQ ID NOs: 5, 6, 1, 2, and 3 respectively). Thisfamily is characterized by an enzyme bearing HNH and RuvC domains. TheRuvC domains of this family have a RuvC_III portion having low homologyto previously described Cas9 family members. Although the initial familymembers have a maximum of 56.8% identity among them, all 6 enzymesexhibit a divergent RuvC_III portion of the RuvC domain and bear thecommon motif of RHHALDAMV (SEQ ID NO:5615), KHHALDAMC (SEQ ID NO:5616),or KHHALDAIC (SEQ ID NO:5617). These motifs are not found in otherdescribed Cas9-like enzymes. The corresponding protein and nucleic acidsequences for these new enzymes and their relevant subdomains arepresented in the sequence listing. Putative tracrRNA sequences wereidentified based on their location relative to the other genes and arepresented as SEQ ID NOs: 5476-5479. The enzyme systems appear to derivefrom the Phylum Verrucomicrobia, the Phylum CandidatusPeregrinibacteria, or the Phylum Candidatus Melainabacteria based on thesequences of 16S rRNAs from genome bins containing the CRISPR systems.The 16S rRNA sequences are presented as SEQ ID NOs: 5592-5596). Adetailed domain-level alignment of the CRISPR system sequences togethercalling out the features described by Shmakov et al. (Mol Cell. 2015Nov. 5; 60(3):385-97), which is entirely incorporated by reference) isdepicted in FIGS. 9A, 9B, 9C, 9D, 9E, 9F, 9G, and 9H.A comparison ofMG1-1, 1-2, and 1-3 versus additional proprietary protein datasetsrevealed additional protein sequences with similar architecture,presented as SEQ NOs: 7-319. These MG1 protein sequences led to thediscovery of additional MG1 motifs as shown in SEQ ID NOs: 5618-5632.

Example 2B.—Discovery of an MG2 Family of CRISPR Systems

Analysis of data from the metagenomic analysis of Example 1 revealed anew cluster of previously undescribed putative CRISPR systems comprisingsix members (MG2-1, MG2-2, MG2-3, MG2-5, and MG2-6). The correspondingprotein and nucleic acid sequences for these new enzymes and exemplarysubdomains are presented as SEQ ID NOs: 320, 322-325. Based on theirlocation relative to the other genes, putative tracrRNA sequences wereidentified in the operon and are presented as SEQ ID NOs: 5490,5492-5494, and 5538. A detailed domain-level alignment of thesesequences versus Cas9 as outlined in Shmakov et al. (Mol Cell. 2015 Nov.5; 60(3):385-97.), is depicted in FIG. 7.

A comparison of MG2-1, MG2-2, MG2-3, MG2-5, and MG2-6 versus additionalproprietary protein datasets revealed additional protein sequences withsimilar architecture, presented as SEQ NOs: 321 and 326-420. Motifscommonly found in MG2 family members are presented as SEQ ID NOs:5631-5638.

Example 2C.—Discovery of an MG3 Family of CRISPR Systems

Analysis of the data from the metagenomic analysis of Example 1 revealeda new previously undescribed putative CRISPR system: MG3-1. Thecorresponding amino acid sequences for this new enzyme and its exemplarysubdomains are presented as SEQ ID NOs: 424, 2245, and 4059. Based onproximity to the other elements in the operon, a putative tracrRNAcontaining sequence was identified and is included as SEQ ID NO: 5498. Adetailed domain-level alignment of the sequence versus Cas9 fromActinomyces naeslundii is depicted in FIG. 8.

A comparison of MG3-1 versus additional proprietary protein datasetsrevealed additional protein sequences with similar architecture,presented as SEQ NOs: 421-423, 425-431.

Example 2D.—Discovery of MG4, 7, 14, 15, 16, 18, 21, 22, 23 Families ofCRISPR Systems

Analysis of the data from the metagenomic analysis of Example 1 revealednew clusters of previously undescribed putative CRISPR systemscomprising 9 families of one member each (MG 4-5, MG7-2, MG14-1, MG15-1,MG16-2, MG18-1, MG21-1, MG22-1, MG23-1). The corresponding protein andnucleic acid sequences for these new enzymes and their exemplarysubdomains are presented as SEQ ID NOs: 432, 669, 678, 930, 1093, 1354,1512, 1656, 1756. Based on proximity to the other elements in theoperon, a putative tracr containing sequence was identified for eachfamily. These sequences are presented in the sequence listing as SEQ IDNOs: 5503-5511, respectively.

A comparison of MG 4-5, MG7-2, MG14-1, MG15-1, MG16-2, MG18-1, MG21-1,MG22-1, MG23-1 versus additional proprietary protein datasets revealedadditional protein sequences with similar architecture, presented as SEQNOs: 433-660, 670-677, 679-929, 931-1092, 1094-1353, 1355-1511,1513-1655, 1657-1755, and 1757-1826. Motifs common to the nucleases ofthese sets of CRISPR systems are presented as SEQ ID NO: 5649 for MG4;SEQ ID NOs: 5650-5667 for MG14; 5668-5675 for MG15; SEQ ID NOs:5676-5678 for MG16; SEQ ID NOs: 5679-5686 for MG18; SEQ ID NOs:5687-5693 and SEQ ID NOs: 5674-5675 for MG21; SEQ ID NOs: 5694-5699 forMG22; and SEQ ID NOs: 5700-5717 for MG23.

Example 3.—Prophetic—Determination of Protospacer-Adjacent Motif

Experiments are performed as in any of the examples in Karvelis et al.Methods. 2017 May 15; 121-122:3-8, which is entirely incorporated byreference herein, to identify the protospacer adjacent motif (PAM)sequence specificity for the novel enzymes described herein to allow foroptimal synthetic sequence targeting.

In one example (in-vivo screen), cells bearing plasmids encoding any ofthe enzymes described herein and protospacer-targeting guide RNA areco-transformed with a plasmid library containing an antibioticresistance gene, and a protospacer sequence flanked by a randomized PAMsequence. Plasmids containing functional PAMs are cleaved by the enzyme,leading to cell death. Deep-sequencing of the enzyme cleavage-resistantplasmid pool isolated from the surviving cells displays a set ofdepleted plasmids that contain functional cleavage-permitting PAMs.

In another example (in vitro screen), PAM library in the form of DNAplasmid or concatemeric repeats is subjected to cleavage by the RNPcomplex (e.g., including the enzyme, tracrRNA and crRNA or the enzymeand hybrid sgRNA) assembled in vitro or in cell lysates. Resulting freeDNA ends from successful cleavage events are captured by adapterligation, followed by the PCR amplification of the PAM-sided products.Amplified library of functional PAMs is subjected to deep sequencing andPAMs licensing DNA cleavage are identified.

Example 4.—Prophetic-Use of Synthetic CRISPR System as Described Hereinin a Mammalian Cell for Genome Editing

DNA/RNA sequences encoding (i) an ORF encoding codon-optimized enzymeunder a cell-compatible promoter with a cell-compatible C-terminalnuclear localization sequence (e.g., SV40 NLS in the case of humancells) and a suitable polyadenylation signal (e.g., TK pA signal in thecase of human cells); and (ii) an ORF encoding an sgRNA (having a 5′sequence beginning with G followed by 20 nt of a complementary targetingnucleic acid sequence targeting genomic DNA followed by a correspondingcompatible PAM identified via Example 3 and a 3′ tracr-binding sequence,a linker, and the tracrRNA sequence) under a suitable Polymerase IIIpromoter (e.g., the U6 promoter in mammalian cells) are prepared. Insome embodiments, these sequences are prepared on the same or separateplasmid vectors, which are transfected via a suitable technique intoeukaryotic cells. In some embodiments, these sequences are prepared asseparate DNA sequences, which are transfected or microinjected intocells. In some embodiments, these sequences are prepared as synthesizedRNAs or in-vitro transcribed RNAs which are transfected or microinjectedinto cells. In some embodiments, these sequences are translated intoproteins and transfected or microinjected into cells.

Whichever transfection method is selected, (i) and (ii) are introducedinto cells. A period of incubation is allowed to pass so that the enzymeand/or sgRNA can be transcribed and/or translated into active form.After the incubation period, genomic DNA in the vicinity of thetargeting sequence is analyzed (e.g., by sequencing). An indel isintroduced into the genomic DNA in the vicinity of the targetingsequence as a result of enzyme-mediated cleavage and non-homologous endjoining.

In some embodiments, (i) and (ii) are introduced into cells with a thirdrepair nucleotide that encodes regions of the genome flanking thecleavage site of sizes 25 bp or larger, which will facilitate homologydirected repair. Containing within these flanking sequences may be asingle base pair mutation, a functional gene fragment, a foreign ornative gene for expression, or several genes composing a biochemicalpathway.

Example 5.—Prophetic-Use of Synthetic CRISPR System as Described HereinIn Vitro

Any of the enzymes described herein are cloned into a suitable E. coliexpression plasmid containing a purification tag and are recombinantlyexpressed in E. coli and purified using the recombinant tag. RNAscomprising a 5′ G followed by a 20 nt targeting sequence and PAMsequence, a tracrRNA binding region of a compatible crRNA, a GAAAlinker, and a compatible tracrRNA are synthesized by suitablesolid-phase RNA synthesis methods. Recombinant enzymes and sgRNA arecombined in a suitable cleavage buffer containing Mg2+ (e.g., 20 mMHEPES pH 7.5, 100 mM KCl, 5 mM MgCl2, 1 mM DTT, 5% glycerol) and thereaction is initiated by introducing a target DNA including a sequencecomplementary to the targeting sequence and PAM sequence. Cleavage ofthe DNA is monitored by a suitable assay (e.g., agarose gelelectrophoresis followed by ethidium bromide staining (or similarlyacting DNA-intercalating agent) and UV visualization).

Example 6.—(General Protocol) PAM Sequence Identification/Confirmationfor the Endonucleases Described Herein

PAM sequences were determined by sequencing plasmids containingrandomly-generated PAM sequences that could be cleaved by putativeendonucleases expressed in an E. coli lysate-based expression system(myTXTL, Arbor Biosciences). In this system, an E. coli codon optimizednucleotide sequence was transcribed and translated from a PCR fragmentunder control of a T7 promoter. A second PCR fragment with a tracrsequence under a T7 promoter and a minimal CRISPR array composed of a T7promoter followed by a repeat-spacer-repeat sequence was transcribed inthe same reaction. Successful expression of the endonuclease and tracrsequence in the TXTL system followed by CRISPR array processing providedactive in vitro CRISPR nuclease complexes.

A library of target plasmids containing a spacer sequence matching thatin the minimal array followed by 8N mixed bases (putative PAM sequences)was incubated with the output of the TXTL reaction. After 1-3 hr, thereaction was stopped and the DNA was recovered via a DNA clean-up kit,e.g., Zymo DCC, AMPure XP beads, QiaQuick etc. Adapter sequences wereblunt-end ligated to DNA with active PAM sequences that had been cleavedby the endonuclease, whereas DNA that had not been cleaved wasinaccessible for ligation. DNA segments comprising active PAM sequenceswere then amplified by PCR with primers specific to the library and theadapter sequence. The PCR amplification products were resolved on a gelto identify amplicons that corresponded to cleavage events. Theamplified segments of the cleavage reaction were also used as templatefor preparation of an NGS library. Sequencing this resulting library,which was a subset of the starting 8N library, revealed the sequenceswhich contain the correct PAM for the active CRISPR complex. For PAMtesting with a single RNA construct, the same procedure was repeatedexcept that an in vitro transcribed RNA was added along with the plasmidlibrary and the tracr/minimal CRISPR array template was omitted. Forendonucleases where NGS libraries were prepared, seqLogo (see e.g.,Huber et al. Nat Methods. 2015 February; 12(2):115-21) representationswere constructed and are presented in FIGS. 27, 38, 29, 30, 31, 32, 33,34, and 35. The seqLogo module used to construct these representationstakes the position weight matrix of a DNA sequence motif (e.g. a PAMsequence) and plots the corresponding sequence logo as introduced bySchneider and Stephens (see e.g. Schneider et al. Nucleic Acids Res.1990 Oct. 25; 18(20):6097-100. The characters representing the sequencein the seqLogo representations have been stacked on top of each otherfor each position in the aligned sequences (e.g. PAM sequences). Theheight of each letter is proportional to its frequency, and the lettershave been sorted so the most common one is on top.

Example 7.—(General Protocol) RNA Folding of tracrRNA and sgRNAStructures

Folded structures of guide RNA sequences at 37° C. were computed usingthe method of Andronescu et al. Bioinformatics. 2007 Jul. 1;23(13):i19-28, which is incorporated by reference herein in itsentirety. Predicted structures of exemplary sgRNAs described herein arepresented in FIGS. 21, 22, 23, 24, 25, and 26.

Example 8.—(General Protocol) In Vitro Cleavage Efficiency of MG CRISPRComplexes

Endonucleases were expressed as His-tagged fusion proteins from aninducible T7 promoter in a protease deficient E. coli B strain. Cellsexpressing the His-tagged proteins were lysed by sonication and theHis-tagged proteins were purified by Ni-NTA affinity chromatography on aHisTrap FF column (GE Lifescience) on an AKTA Avant FPLC (GELifescience). The eluate was resolved by SDS-PAGE on acrylamide gels(Bio-Rad) and stained with InstantBlue Ultrafast coomassie(Sigma-Aldrich). Purity was determined using densitometry of the proteinband with ImageLab software (Bio-Rad). Purified endonucleases weredialyzed into a storage buffer composed of 50 mM Tris-HCl, 300 mM NaCl,1 mM TCEP, 5% glycerol; pH 7.5 and stored at −80° C.

Target DNAs containing spacer sequences and PAM sequences (determinede.g., as in Example 6) were constructed by DNA synthesis. A singlerepresentative PAM was chosen for testing when the PAM had degeneratebases. The target DNAs comprised 2200 bp of linear DNA derived from aplasmid via PCR amplification with a PAM and spacer located 700 bp fromone end. Successful cleavage resulted in fragments of 700 and 1500 bp.The target DNA, in vitro transcribed single RNA, and purifiedrecombinant protein were combined in cleavage buffer (10 mM Tris, 100 mMNaCl, 10 mM MgCl2) with an excess of protein and RNA and incubated for 5minutes to 3 hours, usually 1 hr. The reaction was stopped via additionof RNAse A and incubation at 60 minutes. The reaction was then resolvedon a 1.2% TAE agarose gel and the fraction of cleaved target DNA isquantified in ImageLab software.

Example 9.—(General Protocol) Testing of Genome Cleavage Activity of MGCRISPR Complexes in E. coli

E. coli lacks the capacity to efficiently repair double-stranded DNAbreaks. Thus, cleavage of genomic DNA can be a lethal event. Exploitingthis phenomenon, endonuclease activity was tested in E. coli byrecombinantly expressing an endonuclease and a tracrRNA in a targetstrain with spacer/target and PAM sequences integrated into its genomicDNA.

In this assay, the PAM sequence is specific for the endonuclease beingtested as determined by the methods described in Example 6. sgRNAsequences were determined based upon the sequence and predictedstructure of the tracrRNA. Repeat-anti-repeat pairings of 8-12 bp(generally 10 bp) were chosen, starting from the 5′ end of the repeat.The remaining 3′ end of the repeat and 5′ end of the tracrRNA werereplaced with a tetraloop. Generally, the tetraloop was GAAA, but othertetraloops can be used, particularly if the GAAA sequence is predictedto interfere with folding. In these cases, a TTCG tetraloop was used.

Engineered strains with PAM sequences integrated into their genomic DNAwere transformed with DNA encoding the endonuclease. Transformants werethen made chemocompetent and transformed with 50 ng of single guide RNAseither specific to the target sequence (“on target”), or non-specific tothe target (“non target”). After heat shock, transformations wererecovered in SOC for 2 hrs at 37° C. Nuclease efficiency was thendetermined by a 5-fold dilution series grown on induction media.Colonies were quantified from the dilution series in triplicate.

Example 10a.—(General Protocol) Testing of Genome Cleavage Activity ofMG CRISPR Complexes in Mammalian Cells

To show targeting and cleavage activity in mammalian cells, the MG Caseffector protein sequences were tested in two mammalian expressionvectors: (a) one with a C-terminal SV40 NLS and a 2A-GFP tag, and (b)one with no GFP tag and two SV40 NLS sequences, one on the N-terminusand one on the C-terminus. In some instances, nucleotide sequencesencoding the endonucleases were codon-optimized for expression inmammalian cells.

The corresponding single guide RNA sequence (sgRNA) with targetingsequence attached is cloned into a second mammalian expression vector.The two plasmids are cotransfected into HEK293T cells. 72 hr afterco-transfection of the expression plasmid and a sgRNA targeting plasmidinto HEK293T cells, the DNA is extracted and used for the preparation ofan NGS-library. Percent NHEJ is measured via indels in the sequencing ofthe target site to demonstrate the targeting efficiency of the enzyme inmammalian cells. At least 10 different target sites were chosen to testeach protein's activity.

Example 10b. (General Protocol) Testing of Genome Cleavage Activity ofMG CRISPR Complexes in Mammalian Cells

To show targeting and cleavage activity in mammalian cells, the MG Caseffector protein sequences were cloned into two mammalian expressionvector: (a) one with flanking N and C-terminal SV40 NLS sequences, aC-terminal His tag, and a 2A-GFP tag at the C terminus after the His tag(Backbone 1), and (b) one with flanking NLS sequences and C-terminal Histag but no T2A GFP tag (Backbone 2). In some instances, nucleotidesequences encoding the endonucleases were the native sequence,codon-optimized for expression in E. coli, or codon-optimized forexpression in mammalian cells.

The corresponding single guide RNA sequence (sgRNA) with targetingsequence attached was cloned into a second mammalian expression vector.The two plasmids were cotransfected into HEK293T cells. 72 hr afterco-transfection of the expression plasmid and a sgRNA targeting plasmidinto HEK293T cells, the DNA was extracted and used for the preparationof an NGS-library. Percent NHEJ was measured via indels in thesequencing of the target site to demonstrate the targeting efficiency ofthe enzyme in mammalian cells. About 7-12 different target sites werechosen for testing each protein's activity. An arbitrary threshold of 5%indels was used to identify active candidates.

Example 11.—Characterization of MG1 Family Members

PAM Specificity, tracrRNA sgRNA Validation

The targeted endonuclease activity of MG1 family endonuclease systemswas confirmed using the myTXTL system described in Example 6. In thisassay, PCR amplification of cleaved target plasmids yields a productthat migrates at approximately 170 bp in the gel, as shown in FIGS.17-20. Amplification products were observed for MG1-4 (dual guide: seegel 1, lane 3, single guide: see gel 6 lane 2), MG1-5 (gel 2 lane 10),MG1-6 (dual guide: see gel 5 lane 6, single guide see: gel 6 lane 5),and MG1-7 (dual guide: see gel 3 lane 13, single guide: see gel 3 lane2) (protein SEQ ID NOs: 1-4, respectively). Sequencing the PCR productsrevealed active PAM sequences for these enzymes as shown in Table 2.

TABLE 2 PAM sequence specificities and related data for MG1 enzymesEnzyme Native Synthetic Synthetic protein (dual guide) PAM tracrRNAsgRNA (single guide) (single guide) Enzyme SEQ ID NO PAM SEQ ID NO: SEQID NO: SEQ ID NO: PAM PAM SEQ ID NO: MG1-4 1 nRRRAA 5527 5476 5461 nRRR5512 MG1-5 2 nnnnCC 5528 5477 5462 nnnnYY 5513 MG1-6 3 nnRRAC 5529 54785463 nnRRAY 5514 MG1-7 4 nRRRAA 5530 5479 5464 nRRRAAG 5515

Synthetic single guide RNAs (sgRNAs) were designed based on thesequences and predicted structures of the tracrRNAs and are presented asSEQ ID NOs: 5461-5464. The PAM sequence screen of Example 6 was repeatedwith the sgRNAs. The results of this experiment are also presented inTable 2, which reveals that PAM specificity changed slightly when usingsgRNAs.

Targeted Endonuclease Activity In Vitro

In vitro activity of the MG1-4 endonuclease system (protein SEQ ID NO: 1with sgRNA SEQ ID NO: 5461) on a target DNA with a PAM sequence CAGGAAGGwas verified using the method of Example 8. The single guide sequencereported above (SEQ ID NO: 5461) was used, with varying spacer/targetingsequence lengths from 18-24 nt replacing the Ns of the sequence. Theresults are shown in FIG. 10, wherein the left panel shows a geldemonstrating DNA cleavage by MG1-4 in combination with correspondingsingle guide sgRNAs having different targeting sequence lengths (18-24nt), and the right panel shows the same data quantified as a bar graph.The data demonstrated that targeting sequences from 18-24 nucleotideswere functional with the MG1-4/sgRNA system.

Targeted Endonuclease Activity in Bacterial Cells

In vivo activity of the MG1-4 endonuclease system (protein SEQ ID NO: 1,sgRNA SEQ ID NO: 5461) was tested with the PAM sequence CAGGAAGG as inExample 9. Transformed E. coli were plated in serial dilution, and theresults (showing E. coli serial dilutions in the left panel andquantitated growth in the right panel) are presented in FIG. 11. Asubstantial reduction in the growth of E. coli expressing on targetsgRNA compared to E. coli expressing non-target sgRNA indicates thatgenomic DNA was specifically cleaved by the endonuclease in E. colicells.

Targeted Endonuclease Activity in Mammalian Cells (a)

The method of Example 10 was used to demonstrate targeting and cleavageactivity in mammalian cells. Open reading frames encoding the MG1-4(protein SEQ ID NO: 5527) and MG1-6 (protein SEQ ID NO: 5529) sequenceswere cloned into 2 mammalian expression vectors, one with a C-terminalSV40 NLS and a 2A-GFP tag (E. coli MG-BB) and one with no GFP tag and 2NLS sequences, one on the N-terminus and one on the C-terminus (E. colipMG5-BB). For MG1-6, the open reading frame was additionallycodon-optimized for mammalian expression (SEQ ID NO: 5589) and clonedinto the 2-NLS plasmid backbone (MG-16hs). The results of thisexperiment are shown in FIG. 12. The endonuclease expression vectorswere cotransfected into HEK293T cells with a second vector forexpressing a sgRNA (e.g., SEQ ID NOs: 5512 or 5515) with a tracrsequence specific for the endonuclease and a guide sequence selectedfrom Tables 3-4. 72 hr after co-transfection the DNA was extracted andused for the preparation of an NGS-library. Cleavage activity wasdetected by the appearance of internal deletions (NHEJ remnants)proximal to the sequence of the target site. Percent NHEJ was measuredvia indels in the sequencing of the target site to demonstrate thetargeting efficiency of the enzyme in mammalian cells and is presentedin FIG. 12.

TABLE 3 MG1-4 mammalian targeting sequences MG1-4 Targeting sequenceTarget ID MG1-4 Targeting Sequence SEQ ID NO: Targeted Gene 1aatatgtagctgtttgggaggt 5543 VEGFA 2 ctagggggcgctcggccaccac 5544 VEGFA 3tggctaaagagggaatgggctt 5545 VEGFA 4 cacaccccggctctggctaaag 5546 VEGFA 5tcggaggagccgtggtccgcgc 5547 VEGFA 6 gcggaccacggctcctccgaag 5548 VEGFA 7gtacaaacggcagaagctggag 5549 EMX1 8 gaggaagggcctgagtccgagca 5550 EMX1 9aaggcaaacatcctgataatgg 5551 Apolipoprotein

TABLE 4 MG1-6 mammalian targeting sequences MG1-6 Targeting sequenceTarget ID MG1-6 Targeting Sequence SEQ ID NO: Targeted Gene 1tctttagccagagccggggtgt 5552 VEGFA 2 tggaccccctatttctgacctc 5553 VEGFA 3atgggagcccttcttcttctgc 5554 EMX1 4 tgccacgaagcaggccaatggg 5555 EMX1 5tggtgtctgtttgaggttgcta 5556 HBB-R01 6 gggcaggttggtatcaaggtta 5557HBB-R01 7 aggtgctgacgtaggtagtgct 5558 FANCF 8 gccctacttccgctttcacctt5559 FANCF 9 aatgtatgctggcttttaaggg 5560 IVS40 10 gctcctttggctagggaagtgt5561 IVS40

Targeted Endonuclease Activity in Mammalian Cells (b)

MG-4 target loci were chosen to test locations in the genome with thePAM nRRRAA (SEQ ID NO: 5527). The spacers corresponding to the chosentarget sites were cloned into the sgRNA scaffold in the mammalian vectorsystem Backbone 2 described in Example 10b. The sites are listed inTable 4a below. The activity of MG1-4 at various target sites is shownin Table 4a and FIG. 37

TABLE 4a Activity of MG1-4 at various target sites % NHEJ target IDtarget sequence PAM locus (mean ± std) 1 aatatgtagctgtttgggaggt CAGAAAVEGFA 1.62 ± 1.2  2 ctagggggcgctcggccaccac AGGGAA VEGFA −0.07 ± 0     3tggctaaagagggaatgggctt TGGAAA VEGFA 10.32 ± 2.75  4cacaccccggctctggctaaag AGGGAA VEGFA 0.18 ± 0.53 5 tcggaggagccgtggtccgcgcGGGGAA VEGFA    0 ± 0.03 6 gaagccgagccgagcggagccg CGAGAA VEGFA4.53 ± 1.73 7 gcggaccacggctcctccgaag GAGGAA VEGFA  0.3 ± 0.04 8gtacaaacggcagaagctggag GAAGAA EMX1 0.23 ± 0.1  10Gaaggcaaacatcctgataatgg TGAAAA Apolipoprotein 0.07 ± 0.06

MG1-6 target loci were chosen to test locations in the genome with thePAM nnRRAC (SEQ ID NO: 5529). The spacers corresponding to the chosentarget sites were cloned into the sgRNA scaffold in the mammalian vectorsystem backbone 2 described in Example 10b. The sites are listed inTable 4b below. The activity of MG1-6 at various target sites is shownin Table 4b and FIG. 38.

TABLE 4b Activity of MG1-6 at various target sites. % NHEJ target IDtarget sequence PAM locus (mean ± std) 1 tctttagccagagccggggtgt GCAGACVEGFA 0.385 ± 0.035 2 tggaccccctatttctgacctc CCAAAC VEGFA 0.65 ± 0.62 3atgggagcccttcttcttctgc TCGGAC EMX1 −0.045 ± 0.105  4tgccacgaagcaggccaatggg GAGGAC EMX1 0.96 ± 0.14 5 tggtgtctgtttgaggttgctaGTGAAC HBB −0.09 ± 0.09  6 gggcaggttggtatcaaggtta CAAGAC HBB4.915 ± 0.135 7 aggtgctgacgtaggtagtgct TGAGAC FANCF 3.85 ± 1.86 9aatgtatgctggcttttaaggg GGAAAC IVS40 −0.215 ± 0.105  10gctcctttggctagggaagtgt TAAAAC IVS40 3.03 ± 3.5 

MG1-7 target loci were chosen to test locations in the genome with thePAM nRRRAAG (SEQ ID NO: 5515). The spacers corresponding to the chosentarget sites were cloned into the sgRNA scaffold in the mammalian vectorsystem backbone 2 described in Example 10b. The sites are listed inTable 4c below. The activity of MG1-7 at various target sites is shownin Table 4c and FIG. 39.

TABLE 4c Activity of MG1-7 at various target sites % NHEJ target IDtarget sequence PAM locus (mean ± std) 1 atcaaactgtgagcatcttcag ggaaaagIVS40 0.105 ± 0.88 2 cagcgaatctactcagccagag caggaag IVS40 0.075 ± 0.02 3ctttaacttgtgtgattctgga gaggaag IVS40 −0.25 4 agcttaattagctcctttggctagggaag IVS40 0.545 ± 1.71 5 ccatggtgcatctgactcctga ggagaag HBB0.005 ± 0.01 6 tgcttgagaccgccagaagctc ggaaaag FANCF  0.91 ± 0.38 7cgagaacagcccagaagttgga cgaaaag VEGFA  5.49 ± 0.44 8cggaggagccgtggtccgcgcg ggggaag VEGFA  0.27 ± 0.20 9atgtgtgagaaaaatttaatca taagaag Apolipoprotein −1.85 ± 1.89 10aggtccaagtaaaatttgtctt agaaaag Apolipoprotein −2.17 ± 2.55 11gtacaaacggcagaagctggag gaggaag EMX1 0.775 ± 0.33 12aggaagggcctgagtccgagca gaagaag EMX1  0.01 ± 0.01 13aagggcctgagtccgagcagaa gaagaag EMX1 0.185 ± 0.22

Example 12.—Characterization of MG2 Family Members

PAM Specificity, Tracr RNAs sgRNA Validation

The targeted endonuclease activity of MG2 family members was confirmedin the myTXTL system as described in Example 6. Results of this assayare shown in FIGS. 17-20. In the assay shown in FIGS. 17-20, activeproteins that successfully cleave the library result in a band around170 bp in the gel. Amplification products were observed for MG2-1 (seegel 2 lane 11 and gel 4 lane 6) and MG2-7 (see gel 11 lane 10) (SEQ IDNOs: 320 and 321, respectively). Sequencing the PCR products revealedactive PAM sequences in Table 5 below:

TABLE 5 PAM sequence specificities and related data for MG2 enzymesEnzyme Native Synthetic Synthetic protein (dual guide) PAM tracrRNAsgRNA (single guide) (single guide) Enzyme SEQ ID NO PAM SEQ ID NO: SEQID NO: SEQ ID NO: PAM PAM SEQ ID NO: MG2-1 320 nRCGTA 5531 5490 N/A N/AN/A MG2-7 321 N/A N/A 5491 5465 NNNRTA 5516

Targeted Endonuclease Activity in Bacterial Cells

In vivo activity of the MG2-7 endonuclease system with a sgRNA(endonuclease SEQ ID NO: 321; sgRNA SEQ ID NO: 5465) and an AGCGTAAG PAMsequence was confirmed using the method described in Example 9.Transformed E. coli were plated in serial dilution, and the results(showing E. coli serial dilutions in the left panel and quantitatedgrowth in the right panel) are presented in FIG. 34. A substantialreduction in the growth of E. coli expressing on target sgRNA comparedto E. coli expressing non-target sgRNA indicates that genomic DNA wasspecifically cleaved by the MG1-4 endonuclease in E. coli cells.

Example 13.—Characterization of MG3 Family Members

PAM Specificity, tracrRNA sgRNA Validation

The targeted endonuclease activity of MG3 family members was confirmedusing the myTXTL system as described in Example 6 using tracr sequencesand CRISPR arrays. In this assay, PCR amplification of cleaved targetplasmids yields a product that migrates at approximately 170 bp in thegel, as shown in FIGS. 17-20. Amplification products were observed forMG3-6 (dual guide: see gel 2 lane 8; single guide: see gel 3 lane 3),MG3-7 (dual guide: see gel 2 lane 3, single guide: see gel 3 lane 4),and MG3-8 (dual guide: see gel 9 lane 5) (SEQ ID NOs: 421, 422, and 423,respectively). Sequencing the PCR products revealed active PAM sequencesin Table 6 below:

TABLE 6 Enzyme Native Synthetic Synthetic protein (dual guide) PAMtracrRNA sgRNA (single guide) (single guide) Enzyme SEQ ID NO PAM SEQ IDNO: SEQ ID NO: SEQ ID NO: PAM PAM SEQ ID NO: MG3-6 421 nnRGGTT 5532 55005466 nnGGG 5517 MG3-7 422 nnRnYAY 5533 5501 5467 nnGnTnT 5518 MG3-8 423nnRGGTT 5534 5502 N/A N/A N/A

Synthetic single guide RNAs (sgRNAs) were designed based on thesequences and predicted structures of the tracrRNAs and are presented asSEQ ID NOs: 5466-5467. The PAM sequence screen of Example 6 was repeatedwith the sgRNAs. The results of this experiment are also presented inTable 6, which reveals that PAM specificity changed slightly when usingsgRNAs.

Targeted Endonuclease Activity In Vitro

In vitro activity of the MG3-6 (endonuclease SEQ ID NO: 421) wasverified with the PAM sequence GTGGGTTA using the method of Example 8.The single guide sequence reported above (SEQ ID NO: 5466) was used,with varying spacer/targeting sequence lengths from 18-24 nt replacingthe Ns of the sequence. The results are shown in FIG. 13, wherein thetop panel shows a gel demonstrating DNA cleavage by MG3-6 in combinationwith different sgRNAs having different targeting sequence lengths (18-24nt), and the bottom panel shows the same data quantified as a bar graph.The data demonstrated that targeting sequences from 18-24 nucleotideswere functional with the MG3-6/sgRNA system.

Targeted Endonuclease Activity in Bacterial Cells

In vivo activity of the MG3-7 endonuclease system (protein SEQ ID NO:422; sgRNA SEQ ID NO: 5467) was tested with the PAM sequence TGGACCTGusing the method of Example 9. Transformed E. coli were plated in serialdilution, and the results (showing E. coli serial dilutions in the toppanel and quantitated growth in the bottom panel) are presented in FIG.14. A substantial reduction in the growth of E. coli expressing ontarget sgRNA compared to E. coli expressing non-target sgRNA indicatesthat genomic DNA was being specifically cleaved by the MG3-7endonuclease system.

Targeted Endonuclease Activity in Mammalian Cells (a)

The method of Example 10 was used to demonstrate targeting and cleavageactivity in mammalian cells. Open reading frames encoding MG3-7 (proteinSEQ ID NO: 422) was cloned into 2 mammalian expression vectors, one witha C-terminal SV40 NLS and a 2A-GFP tag (E. coli MG-BB) and one with noGFP tag and 2 NLS sequences, one on the N-terminus and one on theC-terminus (E. coli pMG5-BB). The endonuclease expression vectors werecotransfected into HEK293T cells with a second vector for expressing thesgRNA above with a guide sequence selected from Table 7. The results ofthis experiment are shown in FIG. 12. 72 hr after co-transfection DNAwas extracted and used for the preparation of an NGS-library. Cleavageactivity was detected by the appearance of internal deletions (NHEJremnants) in the vicinity of the target site. Results are presented inFIG. 15.

The target site which were encoded on the sgRNA plasmids are shown inTable 7 below.

TABLE 7 MG3-7 mammalian targeting sequences MG3-7 Targeting sequenceTarget ID MG3-7 Targeting Sequence SEQ ID NO: Targeted Gene 1cccctatttctgacctcccaaa 5563 VEGFA 2 tgtggttccagaaccggaggac 5564 EMX1 3ggccctgggcaggttggtatca 5565 HBB-R01 4 tccttaaacctgtcttgtaacc 5566HBB-R01 5 ctgactcctgaggagaagtctg 5567 HBB-R01 6 tccgagcttctggcggtctcaa5568 FANCF 7 tatcatttcgcggatgttccaa 5569 FANCF 8 tcgggcagagggtgcatcacct5570 Apolipoprotein 9 ataataagcagaacttttagtg 5571 Fibrinogen 10gttttctttagttattaatttc 5572 Fibrinogen

Targeted Endonuclease Activity in Mammalian Cells (b)

MG3-6 target loci were chosen to test locations in the genome with thePAM nnRGGTT (SEQ ID NO: 5532). The spacers corresponding to the chosentarget sites were cloned into the sgRNA scaffold in the mammalian vectorsystem backbone 1 described in Example 10b. The sites are listed inTable 7a below. The activity of MG3-6 at various target sites is shownin Table 7a and FIG. 40.

TABLE 7a Activity of MG3-6 at various target sites target % NHEJ IDtarget sequence PAM locus (mean ± std) target1 gagtgactgaaacttcacagaatagggtt Albumin 9.755 ± 4.67 target2 ttctatttatgagatcaacagc acaggttAlbumin 32.75 ± 7.42 target3 cagatgcaccatggtgtctgtt tgaggt HBB_R012.525 ± 2.13 target5 gaggccctgggcaggttggtat caaggtt HBB_R01 25.16 ± 3.67target6 aggttggtatcaaggttacaag acaggtt HBB_R01 35.27 ± 2.78 target7tcaactttcctggcaacttgcg gtaggtt Apolipoprotei 21.57 ± 5.70 target8aggaacattcagttaagatagt ctaggtt Fibrinogen 7.5675 ± 11.99 target9tttaaattatgaatccatctct aaaggtt Fibrinogen 46.3175 ± 31.46  target10gagtgactgaaacttcacagaa tagggtt Albumin 3.9325 ± 3.53 

MG3-7 target loci were chosen to test locations in the genome with thePAM nnRnTAC (SEQ ID NO: 5718). The spacers corresponding to the chosentarget sites were cloned into the sgRNA scaffold in the mammalian vectorsystems described in Example 10b. The sites are listed in Table 7bbelow. The activity of MG3-7 at various target sites is shown in Table7b and FIG. 41.

TABLE 7b Activity of MG3-7 at various target sites % NHEJ backbone 2% NHEJ target ID target sequence PAM locus (mean ± std) backbone 1 1cccctatttctgacctcccaaa CAGCTAC VEGFA 0.54 ± 0.44 2tgtggttccagaaccggaggac AAAGTAC EMX1 −0.09 ± 0.04  3ggccctgggcaggttggtatca AGGTTAC HBB  8.275 ± 0.3.12 3.87 4tccttaaacctgtcttgtaacc TTGATAC HBB 0.16 ± 0.20 5 ctgactcctgaggagaagtctgCCGTTAC HBB 0.245 ± 0.16  6 tccgagcttctggcggtctcaa GCACTAC FANCF1.84 ± 0.10 3.95 7 tatcatttcgcggatgttccaa TCAGTAC FANCF 0.325 ± 0.68  8tcgggcagagggtgcatcacct GGACTAC Apolipoprotein 13 23.88

MG3-8 target loci were chosen to test locations in the genome with thePAM nnRGGTT (SEQ ID NO: 5534). The spacers corresponding to the chosentarget sites were cloned into the sgRNA scaffold in the mammalian vectorsystem backbone 1 described in Example 10b. The sites are listed inTable 7c below. The activity of MG3-8 at various target sites is shownin Table 7c and FIG. 42.

TABLE 7c Activity of MG3-8 at various target sites % NHEJ target IDtarget sequence PAM locus (mean ± std) target1 gagtgactgaaacttcacagaatagggtt Albumin 10.81 ± 4.26 target2 ttctatttatgagatcaacagc acaggttAlbumin  30.48 ± 11.17 target3 cagatgcaccatggtgtctgtt tgaggt HBB3.3925 ± 2.13  target4 gaagttggtggtgaggccctgg gcaggtt HBB 28.995 ± 11.87target5 gaggccctgggcaggttggtat caaggtt HBB 28.69 ± 1.96 target6aggttggtatcaaggttacaag acaggtt HBB 30.8575 ± 13.24  target7tcaactttcctggcaacttgcg gtaggtt Apolipoprotein 7.9825 ± 3.23  target8aggaacattcagttaagatagt ctaggtt Fibrinogen 20.2925 ± 17.10  target9tttaaattatgaatccatctct aaaggtt Fibrinogen 35.533 ± 25.46 target10gagtgactgaaacttcacagaa tagggtt Albumin   4.9 ± 0.71

Example 13.—Characterization of MG4 Family Members

PAM Specificity, tracrRNA sgRNA Validation

The targeted endonuclease activity of MG4 family endonuclease systemswas confirmed using the myTXTL system as described in Example 6. In thisassay, PCR amplification of cleaved target plasmids yields a productthat migrates at approximately 170 bp in the gel, as shown in FIGS.17-20. Amplification products were observed for, MG4-2 (dual guide: seegel2 lane 9, single guide: see gel 10 lane 7) (SEQ ID NO: 432).Sequencing the PCR products revealed active PAM sequences shown in Table8 below.

TABLE 8 PAM sequence specificities and related data for MG4 enzymesSynthetic Enzyme Synthetic (single guide) protein tracrRNA sgRNA (singleguide) PAM SEQ Enzyme SEQ ID NO SEQ ID NO: SEQ ID NO: PAM ID NO: MG4-5432 5503 5468 nnCCR 5519

Example 14.—Characterization of MG14 Family Members

PAM Specificity, tracrRNA sgRNA Validation

The targeted endonuclease activity of MG14 family members (was confirmedusing the myTXTL system as described in Example 6. In this assay, PCRamplification of cleaved target plasmids yields a product that migratesat approximately 170 bp in the gel, as shown in FIGS. 17-20.Amplification products were observed for MG14-1 (dual guide: see gel 1lane 4, single guide: see gel 3 lane 8) (SEQ ID NO: 678). Sequencing thePCR products revealed active PAM sequence specificities shown in Table 9below.

TABLE 9 PAM sequence specificities and related data for MG14 enzymesNative Synthetic Enzyme (dual guide) (single guide) Synthetic proteinPAM PAM tracrRNA sgRNA PAM (single guide) Enzyme SEQ ID NO determinedSEQ ID NO: SEQ ID NO: SEQ ID NO: determined PAM SEQ ID NO: MG14-1 678NNNNGGTA 5535 5505 5469 NNNGGRTA 5520

Targeted Endonuclease Activity in Bacterial Cells

In vivo activity of the MG14-1 endonuclease system with a sgRNA(endonuclease SEQ ID NO: 678; sgRNA SEQ ID NO: 5469) and a GGCGGGGA PAMsequence was confirmed using the method described in Example 9.Transformed E. coli were plated in serial dilution, and the results(showing E. coli serial dilutions in the left panel and quantitatedgrowth in the right panel) are presented in FIG. 35. A substantialreduction in the growth of E. coli expressing on target sgRNA comparedto E. coli expressing non-target sgRNA indicates that genomic DNA wasspecifically cleaved by the MG1-4 endonuclease in E. coli cells.

Targeted Endonuclease Activity in Mammalian Cells

MG14-1 target loci were chosen to test locations in the genome with thePAM nnnnGGTA (SEQ ID NO: 5535). The spacers corresponding to the chosentarget sites were cloned into the sgRNA scaffold in the mammalian vectorsystem backbone 2 described in Example 10b. The sites are listed inTable 9a below. The activity of MG14-1 at various target sites is shownin Table 9a and FIG. 43.

TABLE 9a Activity of MG14-1 at various target sites % NHEJ target IDtarget sequence PAM locus (mean ± std) 1 cttattaataaaattcaaacat CCTAGGTAAlbumin 0.22 ± 1.04 2 gttggtggtgaggccctgggca GGTTGGTA HBB 0.67 ± 0.16 3ctagttaatggaataaaacatt TTATGGTA Fibrinogen 0.15 4 gcattataatgcaccaaggcttTATAGGTA Fibrinogen 0.2 5 gtggcggggtcccaggtgctga CGTAGGTA FANCF5.33 ± 4.13 6 tgtccgccgccggccggggagg AGGTGGT VEGFA 4.88 ± 0.65 A 7ggtgatgcaccctctgcccgat GCTTGGTA Apolipoprotein −0.35 ± 0.59  8ccacatcaactttcctggcaac TTGCGGTA Apolipoprotein 0.195 ± 0.98  9gctagttaatggaataaaacatt TTATGGTA Fibrinogen 0.6 10gcattataatgcaccaaggctt TATAGGTA Fibrinogen 1.17 ± 1.42

Example 15.—Characterization of MG15 Family Members

PAM Specificity, tracrRNA sgRNA Validation

The targeted endonuclease activity of MG15 family members was confirmedusing the myTXTL system as described in Example 6. In this assay, PCRamplification of cleaved target plasmids yields a product that migratesat approximately 170 bp in the gel, as shown in FIGS. 17-20.Amplification products were observed for MG15-1 (dual guide: see gel 7lane 7, single guide: see gel 3 lane 9) (SEQ ID NO: 930). Sequencing thePCR products revealed active PAM sequence specificities detailed inTable 10 below.

TABLE 10 Enzyme Native Synthetic Synthetic protein (dual guide) PAMtracrRNA sgRNA (single guide) (single guide) Enzyme SEQ ID NO PAM SEQ IDNO: SEQ ID NO: SEQ ID NO: PAM PAM SEQ ID NO: MG15-1 930 nnnnC 5536 55065470 nnnnC 5521

In Vitro Activity

In vitro activity of the MG15-1 endonuclease system (protein SEQ ID NO:930; sgRNA SEQ ID NO:5470) was tested with the PAM sequence GGGTCAAAusing the method of Example 8. The single guide sequence reported above(SEQ ID NO: 5470) was used, with varying spacer/targeting sequencelengths from 18-24 nt (replacing the Ns of the sequence). The resultsare shown in FIG. 16, wherein the top panel shows a gel demonstratingDNA cleavage by MG15-1 in combination with different sgRNAs havingdifferent targeting sequence lengths (18-24 nt), and the bottom panelshows the same data quantified as a bar graph. The data demonstratedthat targeting sequences from 18-24 nucleotides were functional with theMG15-1/sgRNA system.

Targeted Endonuclease Activity in Bacterial Cells

In vivo activity of the MG15-1 endonuclease system with a sgRNA(endonuclease SEQ ID NO: 930; sgRNA SEQ ID NO: 5470) and a GGGTCAAA PAMsequence was confirmed using the method described in Example 9.Transformed E. coli were plated in serial dilution, and the results(showing E. coli serial dilutions in the left panel and quantitatedgrowth in the right panel) are presented in FIG. 35. A substantialreduction in the growth of E. coli expressing on target sgRNA comparedto E. coli expressing non-target sgRNA indicates that genomic DNA wasspecifically cleaved by the MG1-4 endonuclease in E. coli cells.

Example 16.—Characterization of MG16 Family Members

PAM Specificity, tracrRNA sgRNA Validation

The targeted endonuclease activity of MG16 family members was confirmedusing the myTXTL system as described in Example 6. In this assay, PCRamplification of cleaved target plasmids yields a product that migratesat approximately 170 bp in the gel, as shown in FIGS. 17-20.Amplification products were observed for MG16-2 (see gel 11, lane 17)(SEQ ID NO: 1093). Sequencing the PCR products revealed active PAMsequence specificities detailed in Table 11 below.

TABLE 11 PAM sequence specificities and related data for MG16 enzymesSynthetic Enzyme Synthetic (single guide) protein sgRNA (single guide)PAM SEQ Enzyme SEQ ID NO SEQ ID NO: PAM ID NO: MG16-2 1093 5471 nRTnCC5522

Example 17.—Characterization of MG18 Family Members

PAM Specificity, tracrRNA sgRNA Validation

The targeted endonuclease activity of MG18 family members was confirmedusing the myTXTL system as described in Example 6. In this assay, PCRamplification of cleaved target plasmids yields a product that migratesat approximately 170 bp in the gel, as shown in FIGS. 17-20.Amplification products were observed for MG18-1 (dual guide: see gel 9lane 9, single guide: see gel 11 lane 12) (SEQ ID NO: 1354). Sequencingthe PCR products revealed active PAM sequence specificities detailed inTable 12 below.

TABLE 12 PAM sequence specificities and related data for MG18 enzymesEnzyme Native Synthetic Synthetic protein (dual guide) PAM tracrRNAsgRNA (single guide) (single guide) Enzyme SEQ ID NO PAM SEQ ID NO: SEQID NO: SEQ ID NO: PAM PAM SEQ ID NO: MG18-1 1354 nRWART 5537 5508 5472nnnRRT 5523

Targeted Endonuclease Activity in Mammalian Cells

MG18-1 target loci were chosen to test locations in the genome with thePAM nRWART (SEQ ID NO: 5537). The spacers corresponding to the chosentarget sites were cloned into the sgRNA scaffold in the mammalian vectorsystem backbone 1 described in Example 10b. The sites are in Table 12abelow. The activity of MG18-1 at various target sites is shown in Table12a and FIG. 44.

TABLE 12a Activity of MG18-1 at various target sites % NHEJ target IDtarget sequence PAM locus (mean ± std) target2 tgcttattatggacaagtagcaagaaat Fibrinogen 9.003 ± 3.70 target4 tccctgaagatgctcacagttt gatagtIVS40  1.99 ± 2.56 target5 ttttatttctcctgcatatgat gatagt IVS406.303 ± 4.82 target8 tgtggggcaaggtgaacgtgga tgaagt HBB 14.897 ± 2.73 target9 atggtgcatctgactcctgagg agaagt HBB 0.387 ± 0.32 target10agaacagcccagaagttggacg aaaagt VEGFA   4.3 ± 1.29 target11tcctccgaagcgagaacagccc agaagt VEGFA 15.22 ± 2.40

Example 18.—Characterization of MG21 Family Members

PAM Specificity, tracrRNA/sgRNA Validation

The targeted endonuclease activity of MG21 family was confirmed usingthe myTXTL system as described in Example 6. In this assay, PCRamplification of cleaved target plasmids yields a product that migratesat approximately 170 bp in the gel, as shown in FIGS. 17-20.Amplification products were observed for MG21-1 (see gel 11 lane 2) (SEQID NO: 1512). Sequencing the PCR products revealed active PAM sequencespecificities detailed in Table 13 below.

TABLE 13 Synthetic Enzyme Synthetic (single guide) protein sgRNA (singleguide) PAM SEQ Enzyme SEQ ID NO SEQ ID NO: PAM ID NO: MG21-1 1512 5473nnRnR 5524

Example 19.—Characterization of MG22 Family Members

PAM Specificity, tracrRNA sgRNA Validation

The targeted endonuclease activity of MG22 family members was confirmedusing the myTXTL system as described in Example 6. In this assay, PCRamplification of cleaved target plasmids yields a product that migratesat approximately 170 bp in the gel, as shown in FIGS. 17-20. In theassay shown FIGS. 17-20, active proteins that successfully cleave thelibrary result in a band around 170 bp in the gel. Amplificationproducts were observed for MG22-1 (see gel 11 lane 3) (protein SEQ IDNO: 1656). Sequencing the PCR products revealed active PAM sequencespecificities detailed in Table 14 below.

TABLE 14 Native Synthetic Enzyme (dual guide) (single guide) Syntheticprotein PAM PAM tracrRNA sgRNA PAM (single guide) Enzyme SEQ ID NOdetermined SEQ ID NO: SEQ ID NO: SEQ ID NO: determined PAM SEQ ID NO:MG22-1 1656 N/A N/A 5510 5474 nnRCnT 5525

Example 20.—Characterization of MG23 Family Members

PAM Specificity, tracrRNA sgRNA Validation

The targeted endonuclease activity of MG23 family members was confirmedusing the myTXTL system as described in Example 6. In this assay, PCRamplification of cleaved target plasmids yields a product that migratesat approximately 170 bp in the gel, as shown in FIGS. 17-20.Amplification products were observed for MG23-1 (see gel 11 lane 4) (SEQID NO: 1756). Sequencing the PCR products revealed active PAM sequencesspecificities for these enzymes detailed Table 15 below.

TABLE 15 Synthetic Enzyme Synthetic (single guide) protein tracrRNAsgRNA (single guide) PAM SEQ Enzyme SEQ ID NO SEQ ID NO: SEQ ID NO: PAMID NO: MG23-1 1756 5511 5475 nRRA 5526

Systems of the present disclosure may be used for various applications,such as, for example, nucleic acid editing (e.g., gene editing), bindingto a nucleic acid molecule (e.g., sequence-specific binding). Suchsystems may be used, for example, for addressing (e.g., removing orreplacing) a genetically inherited mutation that may cause a disease ina subject, inactivating a gene in order to ascertain its function in acell, as a diagnostic tool to detect disease-causing genetic elements(e.g. via cleavage of reverse-transcribed viral RNA or an amplified DNAsequence encoding a disease-causing mutation), as deactivated enzymes incombination with a probe to target and detect a specific nucleotidesequence (e.g. sequence encoding antibiotic resistance int bacteria), torender viruses inactive or incapable of infecting host cells bytargeting viral genomes, to add genes or amend metabolic pathways toengineer organisms to produce valuable small molecules, macromolecules,or secondary metabolites, to establish a gene drive element forevolutionary selection, to detect cell perturbations by foreign smallmolecules and nucleotides as a biosensor.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. It is not intendedthat the invention be limited by the specific examples provided withinthe specification. While the invention has been described with referenceto the aforementioned specification, the descriptions and illustrationsof the embodiments herein are not meant to be construed in a limitingsense. Numerous variations, changes, and substitutions will now occur tothose skilled in the art without departing from the invention.Furthermore, it shall be understood that all aspects of the inventionare not limited to the specific depictions, configurations or relativeproportions set forth herein which depend upon a variety of conditionsand variables. It should be understood that various alternatives to theembodiments of the invention described herein may be employed inpracticing the invention. It is therefore contemplated that theinvention shall also cover any such alternatives, modifications,variations or equivalents. It is intended that the following claimsdefine the scope of the invention and that methods and structures withinthe scope of these claims and their equivalents be covered thereby.

1. An engineered nuclease system, comprising: (a) an endonucleasecomprising a RuvC_III domain and an HNH domain; and (b) an engineeredguide ribonucleic acid structure configured to form a complex with saidendonuclease comprising: (i) a guide ribonucleic acid sequenceconfigured to hybridize to a target deoxyribonucleic acid sequence; and(ii) a tracr ribonucleic acid sequence configured to bind to saidendonuclease, wherein said endonuclease comprises a sequence having atleast 90% sequence identity to SEQ ID NO:
 421. 2. (canceled) 3.(canceled)
 4. The engineered nuclease system of claim 1, wherein saidendonuclease further comprises an HNH domain.
 5. The engineered nucleasesystem of claim 1, wherein said tracr ribonucleic acid sequencecomprises a sequence with at least 80% sequence identity to about 60 to90 consecutive nucleotides selected from any one of SEQ ID NOs:5476-5511 and SEQ ID NO:
 5538. 6. The engineered nuclease system ofclaim 1, wherein said endonuclease is configured to bind to aprotospacer adjacent motif (PAM) sequence selected from the groupcomprising SEQ ID NOs: 5512-5537.
 7. The engineered nuclease system ofclaim 1, wherein said engineered guide ribonucleic acid structurecomprises at least two ribonucleic acid polynucleotides.
 8. Theengineered nuclease system of claim 1, wherein said engineered guideribonucleic acid structure comprises one ribonucleic acid polynucleotidecomprising said guide ribonucleic acid sequence and said tracrribonucleic acid sequence.
 9. The engineered nuclease system of claim 1,wherein said guide ribonucleic acid sequence is complementary to aprokaryotic, bacterial, archaeal, eukaryotic, fungal, plant, mammalian,or human genomic sequence.
 10. The engineered nuclease system of claim1, wherein said endonuclease comprises one or more nuclear localizationsequences (NLSs) proximal to an N- or C-terminus of said endonuclease.11. The engineered nuclease system of claim 10, wherein said one or moreNLSs comprises a sequence selected from SEQ ID NOs: 5597-5612.
 12. Theengineered nuclease system of claim 1, further comprising a single- ordouble-stranded DNA repair template comprising from 5′ to 3′: a firsthomology arm comprising a sequence of at least 20 nucleotides 5′ to saidtarget deoxyribonucleic acid sequence, a synthetic DNA sequence of atleast 10 nucleotides, and a second homology arm comprising a sequence ofat least 20 nucleotides 3′ to said target deoxyribonucleic acidsequence.
 13. The engineered nuclease system of claim 1, wherein saidengineered nuclease system further comprises a source of Mg²⁺.
 14. Theengineered nuclease system of claim 1, wherein said endonuclease andsaid tracr ribonucleic acid sequence are derived from distinct bacterialspecies within a same phylum.
 15. The engineered nuclease system ofclaim 1, wherein said endonuclease comprises SEQ ID NOs: 1-1826 or avariant thereof having at least 55% identity thereto.
 16. The engineerednuclease system of claim 1, wherein said guide RNA structure comprisesan RNA sequence predicted to comprise a hairpin consisting of a stem anda loop, wherein the stem comprises at least 10base-pairedribonucleotides, and an asymmetric bulge within 4 base pairs of theloop.
 17. The engineered nuclease system of claim 1, wherein said tracrribonucleic acid sequence of said engineered guide ribonucleic acidstructure comprises a hairpin comprising at least 8 base-pairedribonucleotides.
 18. The engineered nuclease system of claim 1, whereinsaid guide ribonucleic acid sequence of said engineered guideribonucleic acid structure is predicted to comprise a hairpin with anuninterrupted base-paired region comprising at least 8 nucleotides of aguide ribonucleic acid sequence and at least 8 nucleotides of a tracrribonucleic acid sequence, and wherein said tracr ribonucleic acidsequence comprises, from 5′ to 3′, a first hairpin and a second hairpin,wherein said first hairpin has a longer stem than said second hairpin.19. (canceled)
 20. (canceled)
 21. (canceled)
 22. (canceled)
 23. Anengineered composition comprising: (a) an endonuclease comprising aRuvC_III domain having at least 90% sequence identity to SEQ ID NO:2242; (b) an engineered guide ribonucleic acid structure configured toform a complex with said endonuclease comprising: (i) a guideribonucleic acid sequence configured to hybridize to a targetdeoxyribonucleic acid sequence; and (ii) a ribonucleic acid sequenceconfigured to bind said endonuclease; and (c) a target DNA sequencecomprising a protospacer adjacent motif sequence comprising 5′-RGG-3,wherein R denotes A or G.
 24. A method of modifying a targetdeoxyribonucleic acid sequence in a cell, comprising introducing to saidcell a composition comprising: (a) an endonuclease comprising a RuvC_IIIdomain and an HNH domain; and (b) an engineered guide ribonucleic acidstructure configured to form a complex with said endonucleasecomprising: (iii) a guide ribonucleic acid sequence configured tohybridize to said target deoxyribonucleic acid sequence; and (iv) atracr ribonucleic acid sequence configured to bind to said endonuclease,wherein said endonuclease comprises a sequence having at least 90%sequence identity to SEQ ID NO: 421.